Allergenic proteins and peptides from Japanese cedar pollen

ABSTRACT

The present invention provides nucleic acid sequences coding for the  Cryptomeria japonica  major pollen allergen Cry j I, Cry j II, Jun s I and Jun v I and fragments or peptides thereof. The present invention also provides purified Cry j I, Cry j II, Jun s I and Jun v I and at least one fragment thereof produced in a host cell transformed with a nucleic acid sequence coding for Cry j I, Cry j II, Jun s I and Jun v I or at least one fragment thereof, and fragments of Cry j I, Cry j II, Jun s I or Jun v I or at least one fragment thereof, and fragments of Cry j I, Cry j II, Jun s I or Jun v I prepared synthetically. Cry j I, Cry j II, Jun s I and Jun v I and fragments thereof are useful for diagnosing, treating, and preventing Japanese cedar pollinosis. The present invention also provides isolated peptides of Cry j I and Cry j II. Peptides within the scope of the invention comprise at least one T cell epitope, or preferably at least two T cell epitopes of Cry j I or Cry j II. The invention also pertains to modified peptides having similar or enhanced therapeutic properties as the corresponding naturally-occurring allergen or portion thereof but having reduced side effects. Methods of treatment or of diagnosis of sensitivity to Japanese cedar pollens in an individual and therapeutic compositions, and multipeptide formulations comprising one or more peptides of the invention are also provided.

RELATED CASES

This application is a continuation of U.S. Ser. No. 09/240,203, filedJan. 29. 1999 (patented)which is a continuation of U.S. Ser. No.08/487,023, filed Jun. 6, 1995 (patented), which is a divisional of U.S.Ser. No. 08/350,225, filed Dec. 6, 1994 (abandoned), which is acontinuation-in-part of U.S. Ser. No. 08/226,248 filed Apr. 8, 1994(abandoned), which is a continuation-in-part of PCT/US93/00139(designating the U.S.) filed Jan. 15, 1993 which is acontinuation-in-part of U.S. Ser. No. 07/938,990 filed Sep. 1. 1992(abandoned) which is a continuation-in-part of U.S. Ser. No. 07/730,452filed Jul. 15, 1991 (abandoned) which is a continuation-in-part of U.S.Ser. No. 07/729,134 filed Jul. 12, 1981 (abandoned). U.S. Ser. No.08/226,248 filed Apr. 8, 1994 (abandoned) is also a continuation-in-partof U.S. Ser. No. 07/975,179 filed Nov. 12, 1992 (abandoned). All of theabove-mentioned cases are hereby incorporated herein by Reference.

BACKGROUND OF THE INVENTION

Genetically predisposed individuals, who make up about 10% of thepopulation, become hypersensitized (allergic) to antigens from a varietyof environmental sources to which they are exposed. Those antigens thatcan induce immediate and/or delayed types of hypersensitivity are knownas allergens. (King, T. P., Adv. Immunol. 23: 77-105, (1976)).Anaphylaxis or atopy, which includes the symptoms of hay fever, asthma,and hives, is one form of immediate allergy. It can be caused by avariety of atopic allergens, such as products of grasses, trees, weeds,animal dander, insects, food, drugs, and chemicals.

The antibodies involved in atopic allergy belong primarily to the IgEclass of immunoglobulins. IgE binds to mast cells and basophils. Uponcombination of a specific allergen with IgE bound to mast cells orbasophils, the IgE may be cross-linked on the cell surface, resulting inthe physiological effects of IgE-antigen interaction. Thesephysiological effects include the release of, among other substances,histamine, serotonin, heparin, a chemotactic factor for eosinophilicleukocytes and/or the leukotrienes, C4, D4, and E4, which causeprolonged constriction of bronchial smooth muscle cells (Hood, L. E. etal. Immunology (2nd ed.), The Benjamin/Cumming Publishing Co., Inc.(1984)). These released substances are the mediators which result inallergic symptoms caused by a combination of IgE with a specificallergen. Through them, the effects of an allergen are manifested. Sucheffects may be systemic or local in nature, depending on the route bywhich the antigen entered the body and the pattern of deposition of IgEon mast cells or basophils. Local manifestations generally occur onepithelial surfaces at the location to at which the allergen entered thebody. Systemic effects can include anaphylaxis (anaphylactic shock),which is the result of an IgE-basophil response to circulating(intravascular) antigen.

Japanese cedar (Sugi; Cryptomeria japonica) pollinosis is one of themost important allergic diseases in Japan. The number of patientssuffering from this disease is on the increase and in some areas, morethan 10% of the population are affected. Treatment of Japanese cedarpollinosis by administration of Japanese cedar pollen extract to effecthyposensitization to the allergen has been attempted. Hyposensitizationusing Japanese cedar pollen extract, however, has drawbacks in that itcan elicit anaphylaxis if high doses are used, whereas when low dosesare used to avoid anaphylaxis, treatment must be continued for severalyears to build up a tolerance for the extract.

The major allergen from Japanese cedar pollen has been purified anddesignated as Sugi basic protein (SBP) or Cry j I. This protein isreported to be a basic protein with a molecular weight of 41-50 kDa anda pI of 8.8. There appear to be multiple isoforms of the allergen,apparently due in part to differential glycosylation (Yasueda et al.(1983) J. Allergy Clin. Immunol. 71: 77-86; and Taniai et al. (1988)FEBS Letters 239: 329-332.

The sequence of the first twenty amino acids at the N-terminal end ofCry j I (SEQ ID NO: 18) and a sixteen amino acid sequence (SEQ ID NO:19) at the carboxy terminus have been determined (Taniai supra).

A second allergen has recently been isolated from the pollen ofCryptomeria japonica (Japanese cedar) (Sakaguchi et al. (1990) Allergy45:309-312). This allergen, designated Cry j II, has been reported tohave a molecular weight of approximately 37 kDa and 45 kDa when assayedon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)under non-reducing and reducing conditions, respectively (Sukaguchi etal., supra). Cry j II was found to have no immunologicalcross-reactivity with Cry j I (Sakaguchi (1990) supra; Kawashima et al.(1992) Int. Arch. Allergy Immunol. 98:110-117). Most patients withJapanese cedar pollinosis were found to have IgE antibodies to both Cryj I and Cry j II, however, 29% of allergic patients had IgE that onlyreacted with Cry j I and 14% of allergic patients had IgE that onlyreacted with Cry j II (Sakaguchi (1990) supra). Isoelectric focusing ofCry j II indicated that this protein has a pI above 9.5, as compared topI 8.6-8.8 for Cry j I (Sakaguchi (1990) supra).

In addition to hyposensitization of Japanese cedar pollinosis patientswith low doses of Japanese cedar pollen extract, U.S. Pat. No.4,939,239, issued Jul. 3, 1990 to Matsuhashi et al., discloses ahyposensitization agent comprising a saccharide covalently linked to aJapanese cedar pollen allergen for hyposensitization of personssensitive to Japanese cedar pollen. This hyposensitization agent isreported to enhance the production of IgG and IgM antibodies, but reduceproduction of IgE antibodies which are specific to the allergen andresponsible for anaphylaxis and allergy. The allergens used in thehyposensitization agent preferably have an NH₂-terminal amino acidsequence ofAsp-Asn-Pro-Ile-Asp-Ser-X-Trp-Arg-Gly-Asp-Ser-Asn-Trp-Ala-Gln-Asn-Arg-Met-Lys-,wherein X is Ser, Cys, Thr, or His (SEQ ID NO: 18). Additionally, Usuiet al. (1990) Int. Arch. Allergy Appl. Immunol. 91: 74-79 reported thatthe ability of a Sugi basic protein (i.e., Cry j I)-pullulan conjugateto elicit the Arthus reaction was markedly reduced, about 1,000 timeslower than that of native Sugi basic protein and suggested that the Sugibasic protein-pullulan conjugate would be a good candidate fordesensitization therapy against cedar pollinosis.

The Cry j I allergen found in Cryptomeria japonica has also been foundto be cross-reactive with allergens in the pollen from other species oftrees, including Cupressus sempervirens. Panzani et al. (Annals ofAllergy 57: 26-30 (1986)) reported that cross reactivity was detectedbetween allergens in the pollens of Cupressus sempervirens andCryptomeria japonica in skin testing, RAST and RAST inhibition. A 50 kDaallergen isolated from Mountain Cedar (Juniperus sabinoides, also knownas Juniperus ashei) has the NH₂-terminal sequence AspAsnProIleAsp (SEQID NO: 25) (Gross et al., (1978) Scand. J. Immunol. 8: 437-441) which isthe same sequence as the first five amino acids of the NH-₂ terminal endof the Cry j I allergen. The Cry j I allergen has also been found to beallergenically cross-reactive with the following species of trees:Cupressus arizonica, Cupressus macrocarpa, Juniperus virginiana,Juniperus communis, Thuya orientalis, and Chamaecyparis obtusa.

Despite the attention Japanese cedar pollinosis allergens have received,definition or characterization of the allergens responsible for itsadverse effects on people is far from complete. Current desensitizationtherapy involves treatment with pollen extract with its attendant risksof anaphylaxis if high doses of pollen extract are administered, or longdesensitization times when low doses of pollen extract are administered.Thus there is a pressing need for the development of compositions andmethods that could be used in detecting sensitivity to Japanese cedarpollen allergens or other immunologically related allergens or intreating sensitivities to such allergens with reduced side effects. Thepresent invention provides materials and methods having one or more ofthese utilities.

SUMMARY OF THE INVENTION

The present invention provides nucleic acid sequences coding for theCryptomeria japonica major pollen allergen Cry j I and fragmentsthereof. The present invention also provides isolated Cry j I or atleast one fragment or peptide thereof produced in a host celltransformed with a nucleic acid sequence coding for Cry j I (SEQ ID NO:I) or at least one fragment thereof and fragments of Cry j I preparedsynthetically. The present invention also provides purified native Cry jI protein.

The present invention further provides Jun v I and Jun s I proteinallergens which are immunologically cross-reactive with Cry j I andfragments of Jun v I and Jun s I produced in a host cell transformedwith a nucleic acid sequence coding for Jun s I or Jun v I respectivelyand fragments of Jun s I and Jun v I prepared synthetically and purifiednative Jun s I and Jun v I. The present invention further providesnucleic acid sequences coding for Jun v I (SEQ ID NO: 94) and Jun s I(SEQ ID NO: 96) and fragments thereof. As used herein, a fragment of thenucleic acid sequence coding for the entire amino acid sequence of Cry jI, Jun s I or Jun v I refers to a nucleotide sequence having fewer basesthan the nucleotide sequence coding for the entire amino acid sequenceof Cry j I, (SEQ ID NO: 2) Jun s I (SEQ ID NO: 95) or Jun v I (SEQ IDNO: 97) and/or mature Cry j I, Jun s I or Jun v I. Cry j I, Jun s I orJun v I and fragments thereof are useful for diagnosing, treating, andpreventing Japanese cedar pollinosis as well as pollinosis caused bypollen from other species of trees wherein such pollen isimmunologically cross-reactive with Japenese cedar pollen allergen.

The present invention also provides nucleic acid sequences coding forthe Cryptomeria japonica major pollen allergen Cry j II (SEQ ID NO: 133)and fragments or peptides thereof. The present invention also providespurified Cry j II (SEQ ID NO: 134) and at least one fragment thereofproduced in a host cell transformed with a nucleic acid sequence codingfor Cry j II or at least one fragment thereof, fragments of Cry j IIprepared synthetically, and purified native Cry j II protein purified tohomogeneity. Cry j II and fragments thereof are useful for diagnosing,treating, and preventing Japanese cedar pollinosis.

As used herein the term “peptides” of the invention include full-lengthprotein or fragments thereof. Peptides of the invention may be producedrecombinantly, by chemical synthesis, or by chemical cleavage of thenative protein allergen. Peptides within the scope of the inventionpreferably comprise at least one T cell epitope, and may comprise atleast two T cell epitopes of Cry j I or Cry j II. The invention furtherprovides peptides comprising at least two regions, each regioncomprising at least one T cell epitope of a Japanese cedar pollenprotein allergen. The invention also provides modified peptides havingsimilar or enhanced therapeutic properties as the corresponding,naturally-occurring allergen or portion thereof, but having reduced sideeffects, as well as modified peptides having improved properties such asincreased solubility and stability. Peptides of the invention alone orin conjunction with other peptides of the invention when administered toa Japanese cedar pollen-sensitive individual or in an individual who issensitive to an allergen cross-reactive with Japanese cedar pollen, arecapable of modifying the allergic response of the individual to aJapanese cedar pollen allergen or an allergen cross-reactive withJapanese cedar pollen such as Jun s I or Jun v I. Methods of treatmentor diagnosis of sensitivity to Japanese cedar pollen or a cross-reactiveallergen in an individual and therapeutic compositions comprising one ormore peptides of the invention are also provided. This invention is moreparticularly described in the appended claims and is described in itspreferred embodiments in the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a graphic representation of affinity purified Cry j I onSuperdex 75 (2.6 by 60 cm) equilibrated with 10 mM sodium acetate (pH5.0) and 0.15 M NaCl;

FIG. 1 b shows an SDS-PAGE (12.5%) analysis of the fractions from themajor peak shown in FIG. 1 a;

FIG. 2 shows a Western blot of isoforms of purified native Cry j Iproteins separated by SDS-PAGE and probed with mAB CBF2;

FIG. 3 is a graphic representation of allergic sera titration ofdifferent purified fractions of purified native Cry j I using plasmafrom a pool of fifteen allergic patients;

FIGS. 4 a-d show the composite nucleic acid sequence (SEQ ID NO: 1 fromthe two overlapping clones JC 71.6 and pUC19JC91a coding for Cry j I.The complete cDNA sequence for Cry j I (SEQ ID NO: 1) is composed of1312 nucleotides, including 66 nucleotides of 5′ untranslated sequence,an open reading frame starting with the codon for an initiatingmethionine of 1122 nucleotides, and a 3′ untranslated region. FIGS. 4a-b also show the deduced amino acid sequence of Cry j I (SEQ ID NO: 2);

FIG. 5 a is a graphic representation of the results of IgE bindingreactivity wherein the coating antigen is soluble pollen extract (SPE)from Japanese cedar pollen;

FIG. 5 b is a graphic representation of the results of IgE bindingreactivity wherein the coating antigen is purified native Cry j I;

FIG. 6 is a graphic representation of the results of a competition ELISAwith pooled human plasma (PHP) from 15 patients wherein the coatingantigen is soluble pollen extract (SPE) from Japanese cedar pollen;

FIG. 7 is a graphic representation of the results of a competition ELISAusing plasma from individual patients (indicated by patient numbers)wherein the coating antigen is soluble pollen extract (SPE) fromJapanese cedar pollen and the competing antigen is purified native Cry jI;

FIG. 8 a is a graphic representation of the results from a directbinding ELISA using plasma from seven individual patients (indicated bypatient numbers) wherein the coating antigen is soluble pollen extract(SPE) from Japanese cedar pollen;

FIG. 8 b is a graphic representation of the results from a directbinding ELISA using plasma from seven individual patients (indicated bypatient numbers) wherein the coating antigen is denatured soluble pollenextract which has been denatured by boiling in the presence of areducing agent, DTT;

FIG. 9 is a graphic representation of a direct ELISA where the wellswere coated with recombinant Cry j I (rCry j I) and IgE binding wasassayed on individual patients;

FIG. 10 a is a graphic representation of the results of a capture ELISAusing pooled human plasma from fifteen patients wherein the wells werecoated with CBF2 (IgG) mAb, PBS was used as a negative antigen control,and the antigen was purified recombinant Cry j I;

FIG. 10 b is a graphic representation of the results of a capture ELISAusing rabbit anti-Amb aI and II, wherein the wells were coated with 20μg/ml CBF2 (IgG), PBS was used as a negative antigen control and theantigen was purified recombinant Cry j I;

FIG. 11 is a graphic representation of a histamine release assayperformed on one Japanese cedar pollen allergic patient using SPE fromJapanese cedar pollen, purified native Cry j I and recombinant Cry j Ias the added antigens;

FIG. 12 is a graphic representation of the results of a T cellproliferation assay using blood from patient #999 wherein the antigen isrecombinant Cry j I protein, purified native Cry j I protein, orselected Cry j I peptides recombinant Amb a 1.1;

FIG. 13 shows various peptides of desired lengths derived from Cry j I(SEQ ID NOs: 26-60);

FIG. 14 is a graphic representation depicting responses of T cell linesfrom twenty-five patients primed in vitro with purified native Cry j Iand analyzed for response to various Cry j I peptides by percent ofresponses (positive) with an S.I of at least two (shown over each bar),the mean stimulation index of positive response for the peptide (shownover each bar in parenthesis) and the positivity index (Y axis);

FIG. 15 is a graphic representation of the results of a direct bindingassay of IgE to certain Cry j I peptides, purified native Cry j I andrCry j I;

FIG. 16 a-c shows the nucleotide sequence (SEQ ID NO: 94) of Jun s I;this sequence is a composite from the two overlapping cDNA clonespUC19JS42e and pUC19JS45a as well as the full-length clone JS53iibcoding for Jun s I; the complete cDNA sequence for Jun s I (SEQ ID NO:94) is composed of 1170 nucleotides, including 25 nucleotides of 5′untranslated sequence, an open reading frame of 1,101 nucleotides, and a3′ untranslated region; FIG. 16 also shows the deduced amino acidsequence of Jun s I (SEQ ID NO; 95);

FIG. 17 shows the nucleotide sequence of Jun v I; this sequence is acomposite from the two overlapping cDNA clones pUC19JV46a andpUC19JV49iia coding for Jun v I; the complete cDNA sequence for Jun v I(SEQ ID NO: 96) is composed of 1278 nucleotides, including 35nucleotides of 5′ untranslated sequence, an open reading frame of 1,110nucleotides, and a 3′ untranslated region; FIG. 17 also show the deducedamino acid sequence of Jun v I (SEQ ID NO: 97);

FIG. 18 shows various peptides of desired lengths derived from Cry j I(SEQ ID NOs: 71-93);

FIGS. 19 a and 19 b show Northern blots of pollen-derived RNA probedwith Cry j cDNA for identification of mRNA capable of encoding Cry j Ior a Cry j I homologue; FIG. 19 a shows RNA from C. japonica (U.S. andJapanese sources), J. sabinoides and J. virginiana probed with Cry j IcDNA; FIG. 19 b shows RNA from J. sabinoides and C. arizonica probedwith the same cDNA; the position of molecular weight standards are shownin each part of the Figure;

FIG. 20 shows various modified peptides of Cry j I (SEQ ID NOs:119-132);

FIG. 21 is a graphic representation depicting regions of T cell linesfrom 26 patients primed in vitro with and analyzed for response tovarious Cry j I peptides and affinity purified Cry j I peptides bypercent of responses;

FIG. 22 is a graphic representation of a direct ELISA assay whereinwells were coated with peptides derived from Cry j I and then assayedfor IgE binding to patient plasma pool A (PHP-A);

FIG. 23 is a graphic representation of a direct ELISA assay whereinwells were coated with peptides derived from Cry j I and then assayedfor IgE binding to patient plasma pool D (PHP-D);

FIG. 24 is a graphic representation of a direct ELISA used to controlfor the presence of Cry j I peptide coating the wells; mouse polyclonalantisera was generated to the peptides

FIG. 25 a shows an SDS-PAGE (12%) analysis of Cry j II undernon-reducing conditions;

FIG. 25 b shows an SDS-PAGE (12%) analysis of Cry j II under reducingconditions.

FIG. 26 shows the results of mono S column chromatography of Cry j IIeluted with a step gradient of NaCl in 10 mM sodium acetate buffer, pH5.0;

FIG. 27 shows an SDS-PAGE (12%) of purified subfractions of Cry j IIanalyzed under reducing conditions;

FIG. 28 a-g shows the nucleic acid sequence (SEQ ID NO: 133) and thededuced amino acid sequence (SEQ ID NO: 134) coding for Cry j II;

FIG. 29 shows the deduced amino acid sequence of Cry j II (SEQ ID NO:134);

FIG. 30 shows the long form and short form NH₂-terminii amino acidsequences of Cry j II determined by protein sequence analysis asdiscussed in Example 14 aligned with the ten amino acid sequence of Cryj II defined by Sakaguchi et al., supra (SEQ ID NOs: 262, 263, 138, 264,and 265);

FIG. 31 is a graphic representation of the results of a direct ELISAassay showing the binding response of the monoclonal antibody 4B11 andseven patients' (Batch 1) plasma IgE to purified Cry j I as the coatingantigen;

FIG. 32 is a graphic representation of a direct ELISA assay showing thebinding response of the monoclonal antibody 4B11, and seven patients'(Batch 1) plasma IgE to purified native Cry j II as the coating antigen;

FIG. 33 is a graphic representation of a direct ELISA assay showing thebinding response of the monoclonal antibody, 4B11, and seven patients'(Batch 1) plasma IgE to recombinant Cry j II (rCry j II) as the coatingantigen;

FIG. 34 is a graphic representation of a direct ELISA assay showing thebinding response of eight patients' (Batch 2) plasma IgE to purifiednative Cry j I;

FIG. 35 is a graphic representation of a direct ELISA assay showing thebinding response of eight patients' (Batch 2) plasma IgE to purifiednative Cry j II;

FIG. 36 is a graphic representation of a direct ELISA assay showing thebinding response of eight patients' (Batch 2) plasma IgE to recombinantCry j II;

FIG. 37 is a graphic representation of a direct ELISA assay showing thebinding response of eight patients' (Batch 3) plasma IgE to purifiednative Cry j I;

FIG. 38 is a graphic representation of a direct ELISA assay showing thebinding response of eight patients' (Batch 3) plasma IgE to purifiednative Cry j II;

FIG. 39 is a graphic representation of a direct ELISA assay showing thebinding response of eight patients' (Batch 3) plasma IgE to recombinantCry j II;

FIG. 40 is a table which summarizes both the MAST scores performed onpatient's plasma samples (Batch 1-3) and the direct ELISA results shownin FIGS. 31-39; a positive response is indicated by a (+) sign and thenumber of positive responses for each antigen is shown at the bottom ofeach column;

FIG. 41 a-b shows various Cry j II peptides (SEQ ID NOs: 185-193);

FIG. 42 is a graphic representation depicting T cell responses to Cry jII peptides Cry j IIA (SEQ ID NO: 185), and Cry j IIB (SEQ ID NO: 186);the mean S. I is shown above each bar (in parentheses) as well as thepercentage of responses, the positivity index (mean S.I. multiplied bypercentage of responses) is the Y axis;

FIG. 43 is a graphic representation depicitng T cell responses to Cry jII peptides Cry j IIC (SEQ ID NO: 187), Cry j IID (SEQ ID NO: 188), Cryj IIE (SEQ ID NO: 189), Cry j IIF (SEQ ID NO: 190); Cry j IIG (SEQ IDNO: 191), Cry j IIH (SEQ ID NO. 192) the mean S.I. is shown above eachbar (in parentheses) as well as the percentage of responses; thepositivity index (mean S.I. multiplied by percentage of responses) isthe Y axis.

FIG. 44 shows various modified Cry j I (SEQ ID NOs: 202-234, 127,235-258, 130, and 259-261);

FIG. 45 is a graphic representation depicting T cell responses tovarious Cry j I peptides. The mean S.I. shown above each bar (inparenthesis) as well as the percentage of responses, the positivityindex (mean S.I. multiplied by percentage of responses) is the Y axis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides nucleic acids encoding Cry j I, the majorallergen found in Japanese cedar pollen as well as nucleic acidsencoding Cry j II, Jun v I, and Jun s I. Preferably, the nucleic acid isa cDNA having a nucleotide sequence which encodes Cry j I, Cry j II, Junv I or Jun s I. The nucleic acid sequence coding for Cry j I shown inFIGS. 4 a and 4 b (SEQ ID NO: 1) contains a 21 amino acid leadersequence from base 66 through base 128. This leader sequence is cleavedfrom the mature protein which is encoded by bases 129 through 1187. Thededuced amino acid sequence of Cry j I is also shown in FIGS. 4 a and 4b (SEQ ID NO: 2). The nucleic acid sequence of the invention codes for aprotein having a predicted molecular weight of 38.5 kDa, with a pI of7.8, and five potential N-linked glycosylation sites. Utilization ofthese glycosylation sites will increase the molecular weight and affectthe pI of the mature protein. There are sequence polymorphisms observedin the nucleic acid sequence of the invention. For example, singleindependent nucleotide substitutions at the codons encoding amino acids38, 51 and 74 (GGA vs. GAA, GTG vs. GCG, and GGG vs. GAG, respectively)of SEQ ID NO: 1 may result in amino acid polymorphisms (G vs. E, V vs.A, and G vs. E, respectively) at these sites. In addition, a singlenucleotide substitution has been detected in one cDNA clone derived fromCryptomeria japonica pollen collected in Japan. This substitution in thecodon for amino acid 60 (TAT vs. CAT) of SEQ ID NO: 1 may result in anamino acid polymorphism (Y vs. H) at this site. Additional silentnucleotide substitutions have been detected. It is expected that thereare additional sequence polymorphisms, and it will be appreciated by oneskilled in the art that one or more nucleotides (up to about 1% of thenucleotides) in the nucleic acid sequence coding for Cry j I may varyamong individual Cryptomeria japonica plants due to natural allelicvariation. Any and all such nucleotide variations and resulting aminoacid polymorphisms are within the scope of the invention. Furthermore,there may be one or more family members of Cry j I. Such family membersare defined as proteins related in function and amino acid sequence toCry j I but encoded by genes at separate genetic loci. These familymembers are also within the scope of this invention.

The nucleic acid sequence coding for Cry j II shown in FIG. 28 (SEQ IDNO: 133) encodes a protein of 514 amino acids. The deduced Cry j IIamino acid sequence is shown in FIGS. 28 and 29. (SEQ ID NO: 134) Directprotein sequence analysis of native purified Cry j II resulted in twoseparate overlapping NH₂-termini sequences, designated Long and Shortcorresponding respectively to amino acids 46 through 89 (SEQ ID NO: 136)and 51 through 89 (SEQ ID NO: 137) of FIGS. 28, 29 and 30. Thefull-length Cry j II sequence contains 20 cysteine residues and threepotential N-linked glycosylation sites with the consensus sequence ofAsn-Xxx-Ser/Thr. The amino acid sequence representing the long form ofCry j II is encoded by the nucleotide sequence extending from bases177-1586 (SEQ ID NO: 139) as shown in FIG. 28, and the amino acidsequence representing the short form of Cry j II is encoded by thenucleotide sequence extending from 192-1586 (SEQ ID NO: 140) as shown inFIG. 28. A host cell transformed with a vector containing the cDNAinsert coding for full-length Cry j II has been deposited with theAmerican Type Culture Collection, ATCC No. 69105.

Fragments of the nucleic acid sequence coding for fragments of Cry j Ior Cry j II or a cross-reactive allergen or equivalents thereof are alsowithin the scope of the invention. The term “nucleic acid” as usedherein is intended to include fragments or equivalents of the nucleicacid. An equivalent of an oligonucleotide sequence is one which is 1) asequence capable of hybridizing to a complementary oligonucleotide towhich the sequence (or corresponding sequence portions) of SEQ ID NO: 1or SEQ. ID. NO.: 133 or fragments thereof hybridizes, or 2) the sequence(or corresponding sequence portion) complementary to SEQ ID NO: 1, orSEQ. ID. NO.: 133 and/or 3) a sequence which encodes a product (e.g., apolypeptide or peptide) having the same functional characteristics ofthe product encoded by the sequence (or corresponding sequence portion)of SEQ ID NO: 1 or SEQ. ID. NO: 133. Whether an equivalent of a nucleicacid must meet one or both criteria will depend on its use (e.g., if itis to be used only as an oligoprobe, it need meet only the first orsecond criteria and if it is to be used to produce a Cry j I or Cry jII, it need only meet the third criterion).

As used herein, the functional equivalent of a peptide includes peptideshaving the same or enhanced ability to bind MHC; peptides capable ofstimulating the same T cell subpopulations; peptides having the same orincreased ability to induce T cell responses such as stimulation(proliferation or cytokine secretion), peptides having the same orincreased ability to induce T cell non-responsiveness or reducedresponsiveness, peptides having reduced IgE binding, and peptides whichelicit minimal IgE synthesis stimulating activity. Minimal IgEstimulating activity refers to IgE synthesis stimulating activity thatis less than the amount of IgE production elicited by purified nativeCry j I, Cry j II, Jun s I or Jun v I.

Preferred nucleic acids encode a peptide having at least about 50%homology to Cry j I (SEQ ID NO: 1) or Cry j II, (SEQ ID NO: 133) morepreferably at least about 60% homology and most preferably at leastabout 70% homology with Cry j I (FIGS. 4 a-b) or Cry j II (FIG. 28).Nucleic acids which encode peptides having at least about 90%, morepreferably at least about 95%, and most preferably at least about 98-99%homology with Cry j I or Cry j II are also within the scope of theinvention. Homology refers to sequence similarity between two peptidesof Cry j I or Cry j II or between two nucleic acid molecules. Homologycan be determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then molecules arehomologous at that position. A degree of homology between sequences is afunction of the number of matching or homologous positions shared by thesequences.

Preferred nucleic acid fragments encode peptides of at least 10 aminoacid residues in length, preferably at least 15 amino acid residues inlength, more preferably at least 20 amino acid residues in length andmost preferably at least 30 amino acid residues in length. Nucleic acidfragments which encode peptides of at least 40 amino acid residues inlength, at least 60 amino acid residues in length, at least 80 aminoacid residues in length, at least 100 amino acid residues in length ormore are also within the scope of this invention.

Nucleic acids within the scope of the invention include those coding forparts of Cry j I (or a cross-reactive allergen such as Jun v I (SEQ IDNO: 96) or Jun s I (SEQ ID NO: 94)) or Cry j II (SEQ ID NO: 133) whichare antigenic i.e. induce an immune response in mammals, preferablyhumans, such as stimulation of minimal amounts of IgE; binding of IgE;eliciting the production of IgG and IgM antibodies; or the eliciting ofa T cell response such as proliferation and/or lymphokine secretionand/or the induction of T cell non responsiveness or reduced T cellresponsiveness.

Nucleotides within the scope of the invention also include those capableof hybridizing with nucleic acid from other plant species for use inscreening protocols to detect allergens that are cross-reactive with Cryj I or Cry j II. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0×SSC at 50° are known to thoseskilled in the art or can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, thesalt concentration in the wash step can be selected from a lowstringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C.

As used herein, a fragment of the nucleic acid sequence coding for Cry jI or Cry j II refers to a nucleotide sequence having fewer nucleotidesthan the nucleotide sequence coding for the entire amino acid sequenceof Cry j I and/or mature Cry j I or Cry j II and/or mature Cry j II.Generally, the nucleic acid sequence coding for the fragment orfragments of Cry j I or Cry j II will be selected from the bases codingfor the mature protein, however, in some instances it may be desirableto select all or a part of a fragment or fragments from the leadersequence portion of the nucleic acid sequence of the invention. Nucleicacid sequence of the invention may also contain linker sequences,modified restriction endonuclease sites and other sequences useful forcloning, expression or purification of Cry j I or Cry j II or fragmentsthereof.

Isolated nucleic acids encoding a Cry j I or Cry j II peptide, asdescribed herein, and having a sequence that differs from the nucleotidesequence shown in FIG. 4 a-b (SEQ ID NO: 1) or FIG. 28 (SEQ ID NO: 133)due to degeneracy in the genetic code are also within the scope of theinvention. Such nucleic acids encode functionally equivalent protein orpeptides (i.e., protein or peptides having at least a portion of theactivity of Cry j I or Cry j II) but differ in sequence from the nucleicacid sequence of FIG. 4 a-b (SEQ ID NO: 1) or FIG. 28 (SEQ ID NO: 133)due to the fact that a number of naturally-occurring amino acids areencoded by more than one nucleotide triplet. Codons that specify thesame amino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the Cry j I or Cry j II protein. However, it isexpected that DNA sequence polymorphisms that do lead to changes in theamino acid sequence of Cry j I or Cry j II will exist within Japanesecedar pollen. One skilled in the art will appreciate that thesevariations in one or more nucleotides (up to about 3-4% of thenucleotides) of the nucleic acids encoding proteins or peptides of Cry jI or Cry j II may exist. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention. Furthermore, there may be one or more isoforms or related,cross-reacting family members of Cry j I or Cry j II. Such isoforms orfamily members are defined as proteins related in function and aminoacid sequence to Cry j I or Cry j II, but are encoded by genes atdifferent loci.

A nucleic acid sequence coding for Cry j I or Cry j II may be obtainedfrom Cryptomeria japonica plants. However, Applicants have found thatmRNA coding for Cry j I was very difficult to obtain from commerciallyavailable Cryptomeria japonica pollen. This inability to obtain mRNAfrom the pollen may be due to problems with storage or transportation ofcommercially available pollen. Applicants have found that fresh pollenand staminate cones are a good source of Cry j I or Cry j II mRNA. Itmay also be possible to obtain the nucleic acid sequence coding for Cryj I or Cry j II from genomic DNA. Cryptomeria japonica is a well-knownspecies of cedar, and plant material may be obtained from wild,cultivated, or ornamental plants. The nucleic acid sequence coding forCry j I or Cry j II may be obtained using the method disclosed herein orany other suitable techniques for isolation and cloning of genes. Thenucleic acid sequence of the invention may be DNA or RNA.

The present invention provides expression vectors and host cellstransformed to express the nucleic acid sequences of the invention. Anucleic acid sequence coding for Cry j I, Cry j II, Jun v I or Jun s Ior at least one fragment thereof may be expressed in bacterial cellssuch as E. coli, insect cells (baculovirus), yeast, or mammalian cellssuch as Chinese hamster ovary cells (CHO). Suitable expression vectors,promoters, enhancers, and other expression control elements may be foundin Sambrook et al. Molecular Cloning: A Laboratory Manual, secondedition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989). Other suitable expression vectors, promoters, enhancers, andother expression elements are known to those skilled in the art.Expression in mammalian, yeast or insect cells leads to partial orcomplete glycosylation of the recombinant material and formation of anyinter- or intra-chain disulfide bonds. Suitable vectors for expressionin yeast include YepSec1 (Baldari et al. (1987) Embo J. 6: 229-234);pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943); JRY88 (Schultz etal. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, SanDiego, Calif.). These vectors are freely available. Baculovirus andmammalian expression systems are also available. For example, abaculovirus system is commercially available (PharMingen, San Diego,Calif.) for expression in insect cells while the pMSG vector iscommercially available (Pharmacia, Piscataway, N.J.) for expression inmammalian cells.

For expression in E. coli, suitable expression vectors include, amongothers, pTRC (Amann et al. (1988) Gene 69: 301-315); pGEX (Amrad Corp.,Melbourne, Australia); pMAL (N.E. Biolabs, Beverly, Mass.); pRIT5(Pharmacia, Piscataway, N.J.); pET-11d (Novagen, Madison, Wis.) Jameelet al., (1990) J. Virol. 64:3963-3966; and pSEM (Knapp et al. (1990)BioTechniques 8: 280-281). The use of pTRC, and pET-11d, for example,will lead to the expression of unfused protein. The use of pMAL, pRIT5pSEM and pGEX will lead to the expression of allergen fused to maltose Ebinding protein (pMAL), protein A (pRIT5), truncated β-galactosidase(PSEM), or glutathione S-transferase (pGEX). When Cry j I, Cry j II,fragment, or fragments thereof is expressed as a fusion protein, it isparticularly advantageous to introduce an enzymatic cleavage site at thefusion junction between the carrier protein and Cry j I, Cry j II orfragment thereof. Cry j I, Cry j II or fragment thereof may then berecovered from the fusion protein through enzymatic cleavage at theenzymatic site and biochemical purification using conventionaltechniques for purification of proteins and peptides. Suitable enzymaticcleavage sites include those for blood clotting Factor Xa or thrombinfor which the appropriate enzymes and protocols for cleavage arecommercially available from, for example, Sigma Chemical Company, St.Louis, Mo. and N.E. Biolabs, Beverly, Mass. The different vectors alsohave different promoter regions allowing constitutive or inducibleexpression with, for example, IPTG induction (PRTC, Amann et al., (1988)supra; pET-11d, Novagen, Madison, Wis.) or temperature induction (pRIT5,Pharmacia, Piscataway, N.J.) . It may also be appropriate to expressrecombinant Cry j I in different E. coli hosts that have an alteredcapacity to degrade recombinantly expressed proteins (e.g. U.S. Pat. No.4,758,512). Alternatively, it may be advantageous to alter the nucleicacid sequence to use codons preferentially utilized by E. coli, wheresuch nucleic acid alteration would not affect the amino acid sequence ofthe expressed protein.

Host cells can be transformed to express the nucleic acid sequences ofthe invention using conventional techniques such as calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,or electroporation. Suitable methods for transforming the host cells maybe found in Sambrook et al. supra, and other laboratory textbooks.

Inducible non-fusion expression vectors include pTrc (Amann et al.,(1988) Gene 69:301-315) and pET11d (Studier et al., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 60-89). While target gene expression relies on host RNApolymerase transcription from the hybrid trp-lac fusion promoter inpTrc, expression of target genes inserted into pET11d relies ontranscription from the T7 gn10-lac 0 fusion promoter mediated bycoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident Xprophage harboring a T7 gn1 under the transcriptional control of thelacUV 5 promoter.

One strategy to maximize recombinant Cry j I, Cry j II, Jun s I, or Junv I expression in E. coli is to express the protein in a host bacteriawith an impaired capacity to proteolytically cleave the recombinantprotein (Gottesman, S., Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990) 119-128).Another strategy would be to alter the nucleic acid sequence of thedesired gene to be inserted into an expression vector so that theindividual codons for each amino acid would be those preferentiallyutilized in highly expressed E. coli proteins (Wada et al., (1992) Nuc.Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences ofthe invention could be carried out by standard DNA synthesis techniques.

The nucleic acids of the invention can also be chemically synthesizedusing standard techniques. Various methods of chemically synthesizingpolydeoxynucleotides are known, including solid-phase synthesis which,like peptide synthesis, has been fully automated in commerciallyavailable DNA synthesizers (See e.g., Itakura et al. U.S. Pat. No.4,598,049; Caruthers et al. U.S. Pat. No. 4,458,066; and Itakura U.S.Pat. Nos. 4,401,796 and 4,373,07 1, incorporated by reference herein).

The present invention also provides a method of producing isolatedJapanese cedar pollen allergen Cry j I or Cry j II or at least onefragment thereof comprising the steps of culturing a host celltransformed with a nucleic acid vector directing expression of anucleotide sequence encoding Japanese cedar pollen allergen Cry j I orCry j II or at least one fragment thereof in an appropriate medium toproduce a mixture of cells and medium containing said Japanese cedarpollen allergen Cry j I or Cry j II; and purifying the mixture toproduce substantially pure Japanese cedar pollen allergen Cry j I, Cry jII or at least one fragment thereof. Host cells transformed with anexpression vector containing DNA coding for Cry j I, Cry j II or atleast one fragment thereof are cultured in a suitable medium for thehost cell. Cry j I or Cry j II peptides can be purified from cellculture medium, host cells, or both using techniques known in the artfor purifying peptides and proteins including ion-exchangechromatography, gel filtration chromatography, ultrafiltration,electrophoresis and immunopurification with antibodies specific for Cryj I or Cry j II or fragments thereof. The terms “isolated” and“purified” are used interchangeably herein and refer to peptidessubstantially free of cellular material or culture medium when producedby recombinant DNA techniques, or chemical precursors or other chemicalswhen synthesized chemically. The present invention also providespurified native Cry j I and Cry j II peptides as discussed in Examples 1and 14 and purified native Jun s I and Jun v I as discussed in Example9.

Another aspect of the invention provides preparations includingtherparutic compositions and formulations comprising Japanese cedarpollen allergen Cry j I (or a cross-reactive allergen such as Jun v I orJun s I) or Cry j II, or at least one fragment thereof, synthesized in ahost cell transformed with a nucleic acid sequence encoding all (or aportion of Japanese cedar pollen allergen Cry j I) or suchcross-reactive allergen or Cry j II, or chemically synthesized, andisolated Japanese cedar pollen allergen Cry j I protein or across-reactive allergen such as Jun v I or Jun s I or Cry j II, or atleast one antigenic fragment thereof produced in a host cell transformedwith a nucleic acid sequence of the invention, or produced by chemicallysynthesis or produced by chemical cleavage of the native allergen. Thepresent invention also provides preparations including therapeuticcompositons and formulations comprising native purified Cry j I and Cryj II proteins or fragments thereof.

Antigenic fragments as defined herein refer to any protein fragment ofCry j I which induces an immune response. As used herein, the term“fragment” of a protein refers to an amino acid sequence having fewerresidues than the entire amino acid sequence of the protein from whichthe fragment is derived. “Specific” antigenic fragments as definedherein refer to any antigenic fragment derived from Cry j I or Cry j IIwith the exception of the Cry j I fragments consisting of amino acids1-20 or 325-340 as shown in FIGS. 4 a-4 b and the exception of Cry j IIfragments which consist of amino acids 55-64 of FIGS. 28 and 30.Specific fragments may also include any fragment of said excepted Cry jI or Cry j II fragments, or any portions of said excepted Cry j I or Cryj II fragments in conjunction with amino acid sequence downstream orupstream of said excepted Cry j I or Cry j II fragments, or inconjunction with any other amino acid sequence.

Antigenic fragments of an allergen from Japanese cedar pollen, or across-reactive allergen such as Jun v I or Jun s I may be obtained, forexample, by screening peptides recombinantly produced from thecorresponding fragment of the nucleic acid sequence of the inventioncoding for such peptides or synthesized chemically using techniquesknown in the art, or fragments may be produced by chemical cleavage ofthe native allergen as is known in the art. The allergen may bearbitrarily divided into fragments of a desired length with no overlapof the peptides, or preferably divided into overlapping fragments of adesired length. The fragments are tested to determine their antigenicity(e.g. the ability of the fragment to induce an immune response).Additionally, antigenic fragments comprising “cryptic epitopes’ may bedetermined. Cryptic epitopes are those determinants in a protein antigenwhich, due to processing and presentation of the native protein antigento the appropriate MHC molecule, are not normally revealed to the immunesystem. However, a peptide comprising a cryptic epitope is capable oftolerizing T cells, and when a subject is primed with the peptide, Tcells obtained from the subject will proliferate in vitro in response tothe peptide or the protein antigen from which the peptide is derived.Peptides which comprise at least one cryptic epitope derived from aprotein antigen are referred to herein as cryptic peptides. To confirmthe presence of cryptic epitopes in the above-described assay,antigen-primed T cells are cultured in vitro in the presence of eachpeptide separately to establish peptide-reactive T cell lines. A peptideis considered to comprise at least one cryptic epitope if a T cell linecan be established with a given peptide and T cells are capable ofproliferation upon challenge with the peptide and the protein antigenfrom which the peptide is derived.

If fragments of Cry j I or Cry j II are to be used for therapeuticpurposes, then the fragments of Cry j I or Cry j II which are capable ofeliciting a T cell response such as stimulation (i.e., proliferation orlymphokine secretion) and/or are capable of inducing T cellnon-responsiveness are particularly desirable and fragments of Japanesecedar pollen which have minimal IgE stimulating activity are alsodesirable. Additionally, for therapeutic purposes, it is preferable touse isolated Japanese cedar pollen allergens, e.g. Cry j I or Cry j II,or fragments thereof or a specific fragment thereof which are capable ofeliciting T cell responses and which do not bind IgE specific forJapanese cedar pollen or bind such IgE to a substantially lesser extent(i.e., at least 100-fold less binding and more preferably at least1,000-fold less binding) than the purified native Japanese cedar pollenallergen binds such IgE. If the isolated Japanese cedar pollen allergenor fragment or fragments thereof bind IgE, it is preferable that suchbinding does not result in the release of mediators (e.g. histamines)from mast cells or basophils. Furthermore, if Jun v I or Jun s I are tobe used for therapeutic purposes, it is preferable to use Juniperuspollen allergens, e.g. Jun v I or Jun s I or a fragment thereof whichare capable of eliciting T cell responses and which do not bind IgEspecific for pollen from the species Juniperus or bind such IgE to asubstantially lesser extent (as defined above) than the purified nativeJuniperus pollen allergen binds such IgE. If the isolated Jun v I or Juns I or fragment or fragments thereof bind IgE, it is preferable thatsuch binding does not result in the release of mediators (e.g.histamines) from mast cells or basophils.

Screening peptides of Cry j I or Cry j II as described herein can beaccomplished using one or more of several different assays. For example,in vitro, Cry j I or Cry j II T cell stimulatory activity is assayed bycontacting a protein or peptide known or suspected to be from Cry j I orCry j II with an antigen presenting cell which presents appropriate MHCmolecules in a T cell culture. Presentation of a peptide of Cry j I orCry j II in association with appropriate MHC molecules to T cells inconjunction with the necessary costimulation has the effect oftransmitting a signal to the T cell that induces the production ofincreased levels of cytokines, particularly of interleukin-2 andinterleukin-4. The culture supernatant can be obtained and assayed forinterleukin-2 or other known cytokines. For example, any one of severalconventional assays for interleukin-2 can be employed, such as the assaydescribed in Proc. Natl. Acad. Sci USA, 86:1333 (1989) the pertinentportions of which are incorporated herein by reference. A kit for anassay for the production of interferon is also available from GenzymeCorporation (Cambridge, Mass.).

A common assay for T cell proliferation entails measuring tritiatedthymidine incorporation. The proliferation of T cells can be measured invitro by determining the amount of ³H-labeled thymidine incorporatedinto the replicating DNA of cultured cells. Therefore, the rate of DNAsynthesis and, in turn, the rate of cell division can be quantified.

In another embodiment, a Cry j I or Cry j II peptide is screened for theability to reduce T cell responsiveness. The ability of a peptide knownto stimulate T cells, to inhibit or completely block the activity ofpurified native Cry j I or Cry j II or portion thereof and induce astate of T cell nonresponsiveness or reduced T cell responsiveness, canbe determined using subsequent attempts at stimulation of the T cellswith antigen presenting cells that present native Cry j I or Cry j IIfollowing exposure to a Cry j I or Cry j II peptide activity. If the Tcells are unresponsive to the subsequent activation attempts, asdetermined by interleukin-2 synthesis and T cell proliferation, a stateof nonresponsiveness has been induced. See, e.g., Gimmi, et al. (1993)Proc. Natl. Acad. Sci USA, 90:6586-6590; and Schwartz (1990) Science,248:1349-1356, for assay systems that can be used as the basis for anassay in accordance with the present invention.

In yet another embodiment, peptides of Cry j I or Cry j II or of animmunologically related allergen such as Jun s I or Jun v I, areidentified by IgE binding activity. For therapeutic purposes, peptidesof the invention preferably do not bind IgE specific for Japanese cedarpollen allergen, or bind such IgE to a substantially lesser extent (e.g.at least 100 fold less and more preferably, at least 1000 fold lessbinding) than the corresponding purified native Cry j I or Cry j IIallergen binds IgE. If a peptide of the invention is to be used as adiagnostic reagent, it is not necessary that the peptide or protein havereduced IgE binding activity compared to the native Cry j I or Cry j IIallergen. IgE binding activity of peptides can be determined by, forexample, an enzyme linked immunosorbent assay (ELISA) using, forexample, sera obtained from a subject, (i.e., an allergic subject) thathas been previously exposed to the native Cry j I or Cry j II allergen.Briefly, a peptide to be tested is coated onto wells of a microtiterplate. After washing and blocking the wells, antibody solutionconsisting of the plasma of an allergic subject who has been exposed tothe peptide being tested or the protein from which it was derived isincubated in the wells. The plasma is generally depleted of IgG beforeincubation. A labeled secondary antibody is added to the wells andincubated. The amount of IgE binding is then quantified and compared tothe amount of IgE bound by a purified native Cry j I or Cry j IIprotein. Alternatively, the binding activity of a peptide can bedetermined by Western blot analysis. For example, a peptide to be testedis run on a polyacrylamide gel using SDS-PAGE. The peptide is thentransferred to nitrocellulose and subsequently incubated with sera froman allergic subject. After incubation with the labeled secondaryantibody, the amount of IgE bound is then determined and quantified.

Another assay which can be used to determine IgE binding activity of apeptide is a competition ELISA assay. Briefly, an IgE antibody pool isgenerated by combining plasma from Japanese cedar pollen allergicsubjects that have been shown by direct ELISA to have IgE reactive withnative Cry j I or Cry j II. This pool is used in ELISA competitionassays to compare IgE binding to native Cry j I or Cry j II to thepeptide tested. IgE binding for the native Cry j I or Cry j II proteinand the peptide being tested is determined and quantified.

If a peptide of Cry j I or Cry j II binds IgE, and is to be used as atherapeutic agent, it is preferable that such binding does not result inthe release of mediators (e.g. histamines) from mast cells or basophils.To determine whether a peptide which binds IgE results in the release ofmediators, a histamine release assay can be performed using standardreagents and protocols obtained for example, from Amac, Inc. (Westbrook,Me.). Briefly, a buffered solution of a peptide to be tested is combinedwith an equal volume of whole heparinized blood from an allergicsubject. After mixing and incubation, the cells are pelleted and thesupernatants are processed an analyzed using a radioimmunoassay todetermine the amount of histamine released.

Isolated protein allergens from Japanese cedar pollen or preferredantigenic fragments thereof, when administered to a Japanese cedarpollen-sensitive individual, or an individual allergic to an allergencross-reactive with Japanese cedar pollen allergen, such as allergenfrom the pollen of Juniperus virginiana or Juniperus sabinoides etc.(discussed previously) arc capable of modifying the allergic response ofthe individual to Japanese cedar pollen or such cross-reactive allergenof the individual, and preferably are capable of modifying the B-cellresponse, T-cell response or both the B-cell and the T-cell response ofthe individual to the allergen. As used herein, modification of theallergic response of an individual sensitive to a Japanese cedar pollenallergen or cross-reactive allergen can be defined as non-responsivenessor diminution in symptoms to the allergen, as determined by standardclinical procedures (See e.g. Varney et al, British Medical Journal,302:265-269 (1990)) including diminution in Japanese cedar polleninduced asthmatic symptoms. As referred to herein, a diminution insymptoms includes any reduction in allergic response of an individual tothe allergen after the individual has completed a treatment regimen witha peptide or protein of the invention. This diminution may be subjective(i.e. the patient feels more comfortable in the presence of theallergen). Diminution in symptoms can be determined clinically as well,using standard skin tests as is known in the art.

Isolated Cry j I or Cry j II protein or fragments thereof are preferablytested in mammalian models of Japanese cedar pollinosis such as themouse model disclosed in Tamura et al. (1986) Microbiol. Immunol. 30:883-896, or U.S. Pat. No. 4,939,239; or the primate model disclosed inChiba et al. (1990) Int. Arch. Allergy Immunol. 93: 83-88. Initialscreening for IgE binding to the protein or fragments thereof may beperformed by scratch tests or intradermal skin tests on laboratoryanimals or human volunteers, or in in vitro systems such as RAST(radioallergosorbent test), RAST inhibition, ELISA assay,radioimmunoassay (RIA), or histamine release (see Examples 7 and 8).

Antigenic fragments of the present invention which have T cellstimulating activity, and thus comprise at least one T cell epitope areparticularly desirable. Specific peptides of Cry j I and Cry j IIcomprising at least one epitope are discussed later. T cell epitopes arebelieved to be involved in initiation and perpetuation of the immuneresponse to a protein allergen which is responsible for the clinicalsymptoms of allergy. These T cell epitopes are thought to trigger earlyevents at the level of the T helper cell by binding to an appropriateHLA molecule on the surface of an antigen presenting cell andstimulating the relevant T cell subpopulation. These events lead to Tcell proliferation, lymphokine secretion, local inflammatory reactions,recruitment of additional immune cells to the site, and activation ofthe B cell cascade leading to production of antibodies. One isotype ofthese antibodies, IgE, is fundamentally important to the development ofallergic symptoms and its production is influenced early in the cascadeof events, at the level of the T helper cell, by the nature of thelymphokines secreted. A T cell epitope is the basic element or smallestunit of recognition by a T cell receptor, where the epitope comprisesamino acids essential to receptor recognition. Amino acid sequenceswhich mimic those of the T cell epitopes and which modify the allergicresponse to protein allergens are within the scope of this invention.

Exposure of Japanese cedar pollen patients to isolated peptides of thepresent invention or to the antigenic fragments of the present inventionwhich comprise at least one T cell epitope and are derived from proteinallergens, in a non-immunogenic form, may cause T cellnon-responsiveness of appropriate T cell subpopulations such that theybecome unresponsive to the protein allergen and do not participate instimulating an immune response upon such exposure or reduced T cellresponsiveness. In addition, administration of a protein allergen of theinvention or an antigenic fragment of the present invention whichcomprises at least one T cell epitope may modify the lymphokinesecretion profile as compared with exposure to the naturally-occurringprotein allergen or portion thereof (e.g. result in a decrease of IL-4and/or an increase in IL-2). Furthermore, exposure to such proteinallergen or antigenic fragment of such protein allergen may influence Tcell subpopulations which normally participate in the response to theallergen such that these T cells are drawn away from the site(s) ofnormal exposure to the allergen (e.g., nasal mucosa, skin, and lung)towards the site(s) of therapeutic administration of the fragment orprotein allergen. This redistribution of T cell subpopulations mayameliorate or reduce the ability of an individual's immune system tostimulate the usual immune response at the site of normal exposure tothe allergen, resulting in a dimunution in allergic symptoms.

The isolated Cry j I and/or Cry j II peptides including antigenicfragments derived therefrom can be used in methods of diagnosing,treating and preventing allergic reactions to Japanese cedar pollenallergen or a cross reactive protein allergen. Thus the presentinvention provides therapeutic compositions comprising isolated Japanesecedar pollen allergen Cry j I or Cry j II or at least one antigenicfragment of specific antigenic fragment thereof produced in a host celltransformed to express Cry j I or Cry j II, or at least one antigenicfragment thereof, and a pharmaceutically acceptable carrier or diluent.The therapeutic compositions of the invention may also comprise Cry j Ior Cry j II or at least one antigenic fragment thereof which may beprepared synthetically or by chemical cleavage of the allergen, and apharmaceutically acceptable carrier or diluent. Administration of thetherapeutic compositions of the present invention to an individual to bedesensitized can be carried out using known techniques. Cry j I or Cry jII peptide may be administered to an individual in combination with, forexample, an appropriate diluent, a carrier and/or an adjuvant orincomplete adjuvant. Pharmaceutically acceptable diluents include salineand aqueous buffer solutions. Pharmaceutically acceptable carriersinclude polyethylene glycol (Wie et al. (1981) Int. Arch. Allergy Appl.Immunol. 64:84-99) and liposomes (Strejan et al. (1984) J. Neuroimmunol7: 27).

The therapeutic compositions of the invention are administered toJapanese cedar pollen-sensitive individuals or individuals sensitive toan allergen which is immunologically cross-reactive with Japanese cedarpollen allergen (i.e. Juniperus virginiana, or Juniperus sabinoides,etc.). For purposes of inducing T cell non-responsiveness, therapeuticcompositions of the invention are preferably administered innon-immunogenic form, e.g. which does not contain adjuvant. While notintending to be limited to any theory, it is believed that T cell nonresponsivness or reduced T cell responsiveness is induced as a result ofnot providing a “second signal” Briefly, it is believed that stimulationof T cells requires two types of signals, the first is the recognitionby the T cell via the T cell receptor of appropriate MHC-associatedprocessed antigens on antigen presenting class (APCs) and the secondtype of signal is referred to as a “second signal” or “costimulatorysignals” which may be provided by certain competent APCs. When acomposition of the invention is administered without adjuvant, it isbelieved that competent APCs which are capable of producing the secondsignal or costimulatory signal are not engaged in the stimulation ofappropriate T cells therefore resulting in T cell non responsiveness orreduced T cell responsiveness. In addition, there are a number ofantibodies or other reagents capable of blocking the delivery ofcostimulatory signals such as the “second signal” which include, but arenot limited to B7 (including B7-1, B7-2, and BB-1), CD28, CTLA4, CD40CD40L CD54 and CD11a/18 (Jenkins and Johnson, Current Opinion inImmunology, 5:361-367 (1993), and Clark and Ledbetter, Nature,367:425-428 (1994)) Thus, a peptide of the invention may be administeredin nonimmunogenic form as discussed above, in conjunction with a reagentcapable of blocking costimulatory signals such that the level of T cellnonresponsiveness is enhanced.

Administration of the therapeutic compositions of the present inventionto an individual to be desensitized can be carried out using knownprocedures at dosages and for periods of time effective to reducesensitivity (i.e., reduce the allergic response) of the individual tothe allergen. Effective amounts of the therapeutic compositions willvary according to factors such as the degree of sensitivity of theindividual to Japanese cedar pollen, the age, sex, and weight of theindividual, and the ability of the protein or fragment thereof to elicitan antigenic response in the individual. The active compound (i.e.,protein or fragment thereof) may be administered in a convenient mannersuch as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, the activecompound may be coated within a material to protect the compound fromthe action of enzymes, acids and other natural conditions which mayinactivate the compound.

For example, preferably about 1 μg-3 mg and more preferably from about20-500 μg of active compound (i.e., protein or fragment thereof) perdosage unit may be administered by injection. Dosage regimen may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

To administer protein or peptide by other than parenteraladministration, it may be necessary to coat the protein with, orco-administer the protein with, a material to prevent its inactivation.For example, protein or fragment thereof may be administered in anadjuvant, co-administered with enzyme inhibitors or in liposomes. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol. 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethyline glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions of dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glyceral,propylene glycol, and liquid polyetheylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as licithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thirmerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol and sorbitol or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about,including in the composition, an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating activecompound (i.e., protein or peptide) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile indectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingwhich yields a powder of the active ingredient (i.e., protein orpeptide) plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

When protein or peptide thereof is suitably protected, as describedabove, the protein may be orally administered, for example, with aninert diluent or an assimilable edible carrier. The protein and otheringredients may also be enclosed in a hard or soft shell gelatincapsule, compressed into tablets, or incorporated directly into theindividual's diet. For oral therapeutic administration, the activecompound may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the composition and preparations may, of course, bevaried and may conveniently be between about 5 to 80% of the weight ofthe unit. The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage will be obtained. Preferredcompositions or preparations according to the present invention areprepared so that an oral dosage unit contains between from about 10 μgto about 200 mg of active compound.

The tablets, troclics, pills, capsules and the like may also contain thefollowing: a binder such as gum gragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup or elixir may contain the active compound, sucrose as a sweeteningagent, methyl and propylparabens as preservative, a dye and flavoringsuch as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompound may be incorporated into sustained-release preparations andformulations.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like. The useof such media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the therapeuticcompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit from as used herein refers to physically discrete unitssuited as unitary dosages for the mammalian subjects to be treated; eachunit containing a predetermined quantity of active compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the novel dosageunit forms of the invention are dictated by and directly dependent on(a) the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

The Cry j I cDNA (SEQ ID NO: 1) or the Cry j II cDNA (SEQ ID NO: 133)(or the mRNAs from which they were transcribed) or a portion thereof canbe used to identify similar sequences in any variety or type of plantand thus, to identify or “pull out” sequences which have sufficienthomology to hybridize to the Cry j I or Cry j II cDNA or mRNA or portionthereof, for example, DNA from allergens of Juniperus virginiana,Juniperus sabinoides etc., under conditions of low stringency. Thosesequences which have sufficient homology (generally greater than 40%)can be selected for further assessment using the method describedherein. Homology can be determined as discussed previously.Alternatively, high stringency conditions can be used. In this manner,DNA of the present invention can be used to identify, in other types ofplants, preferably related families, genera, or species such asJuniperus, or Cupressus, sequences encoding polypeptides having aminoacid sequences similar to that of Japanese cedar pollen allergen Cry j Ior Cry j II, and thus to identify allergens in other species. Thus, thepresent invention includes not only Cry j I or Cry j II, but also otherallergens encoded by DNA which hybridizes to DNA of the presentinvention. The invention further includes isolated allergenic proteinsor fragments thereof that are immunologically related to Cry j I orfragments thereof, such as by antibody cross-reactivity wherein theisolated allergenic proteins or fragments thereof are capable of bindingto antibodies specific for the protein and peptides of the invention, orby T cell cross-reactivity wherein the isolated allergenic proteins orfragments thereof are capable of stimulating T cells specific for theprotein and peptides of this invention.

Proteins or peptides encoded by the cDNA of the present invention can beused, for example as “purified” allergens. Such purified allergens areuseful in the standardization of allergen extracts which are currentlykey reagents for the clinical diagnosis and treatment of Japanese cedarpollinosis.

Another aspect of the invention pertains to an antibody specificallyreactive with Cry j I or Cry j II, or a fragment thereof. The antibodiesof this invention can be used to standardize allergen extracts or toisolate the naturally-occurring or native form of Cry j I or Cry j II.For example, by using proteins or fragments thereof based on the cDNAsequence of Cry j I or Cry j II, anti-proteinlanti-peptide antisera ormonoclonal antibodies can be made using standard methods. A mammal suchas a mouse, a hamster or rabbit can be immunized with an immunogenicform of such protein or an antigenic fragment which is capable ofeliciting an antibody response. Techniques for conferring immunogenicityon a protein or peptide include conjugation to carriers or othertechniques well known in the art. Cry j I or Cry j II protein orfragments thereof can be administered in the presence of adjuvant. Theprogress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassays can beused with the immunogen as antigen to assess the levels of antibodies.

Following immunization, anti-Cry j I or Cry j II antisera can beobtained and, if desired, polyclonal anti-Cry j I or Cry j II antibodiesisolated from the serum. To produce monoclonal antibodies,antibody-producing cells (lymphocytes) can be harvested from animmunized animal and fused by standard somatic cell fusion procedureswith immortalizing cells such as myeloma cells to yield hybridoma cells.Such techniques are well known in the art, for example the hybridomatechnique originally developed by Kohler and Milstein, (Nature (1975)256:495-497) as well as other techniques such as the human B cellhybridoma technique (Kozbar et al., Immunology Today (1983) 4:72) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., Monoclonal Antibodies and Cancer Therapy (1985) Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with Cry j I or Cry j IIand the monoclonal antibodies isolated.

The term antibody as used herein is intended to include fragmentsthereof which are also specifically reactive with Cry j I or Cry j II.Antibodies can be fragmented using conventional techniques and thefragments screened for utility in the same manner as described above forwhole antibodies. For example, F(ab′)₂ fragments can be generated bytreating antibody with pepsin. The resulting F(ab′)₂ fragment can betreated to reduce disulfide bridges to produce Fab′ fragments. Theantibody of the present invention is further intended to includebispecific and chimeric molecules having an anti-Cry j I or Cry j IIportion.

Another aspect of this invention provides T cell clones and soluble Tcell receptors specifically reactive with Cry j I or Cry j II or afragment thereof. Monoclonal T cell populations (i.e., T cellsgenetically identical to one another and expressing identical T cellreceptors) can be derived from an individual sensitive to Cry j I or Cryj II, followed by repetitive in vitro stimulation with Cry j I or Cry jII in the presence of MHC-matched antigen-presenting cells. Single Cry jI or Cry j II MRC responsive cells can then be cloned by limitingdilution and permanent lines expanded and maintained by periodic invitro restimulation. Alternatively, Cry j I or Cry j II specific T-Thybridomas can be produced by a technique similar to B cell hybridomaproduction. For example, a mammal, such as a mouse can be immunized withCry j I or Cry j II or fragments thereof, T cells from the mammal can bepurified and fused with an autonomously growing T cell tumor line. Fromthe resulting hybridomas, cells responding to Cry j I or Cry j II orfragments thereof are selected and cloned. Procedures for propagatingmonoclonal T cell populations are described in Cellular and MolecularImmunology (Abul K. Abbas et al. ed.), W.B. Saunders Company,Philadelphia, Pa. (1991) page 139. Soluble T cell receptors specificallyreactive with Cry j I or Cry j II or fragments thereof can be obtainedby immunoprecipitation using an antibody against the T cell receptor asdescribed in Immunology: A Synthesis (Second Edition), Edward S. Golubet al., ed., Sinauer Associates, Inc., Sunderland, Mass. (1991) pages366-269.

T cell clones specifically reactive with Cry j I or Cry j II orfragments thereof can to be used to isolate and molecularly clone thegene encoding the relevant T cell receptor. In addition, a soluble Tcell receptor specifically reactive with Cry j I or Cry j II orfragments thereof can be used to interfere with or inhibitantigen-dependent activation of the relevant T cell subpopulation, forexample, by administration to an individual sensitive to Japanese Cedarpollen. Antibodies specifically reactive with such a T cell receptor canbe produced according to the techniques described herein. Suchantibodies can be used to block or interfere with the T cell interactionwith peptides presented by MHC.

Through use of the peptides of the present invention, preparations ofconsistent, well-defined composition and biological activity can be madeand administered for therapeutic purposes (e.g. to modify the allergicresponse of a Japanese cedar sensitive individual to pollen of suchtrees). Administration of such peptides or protein may, for example,modify B-cell response to Cry j I or Cry j II, T-cell response to Cry jI or Cry j II or both responses. Isolated peptides can also be used tostudy the mechanism of immunotherapy of Cryptomeria japonica allergy andto design modified derivatives or analogues useful in immunotherapy.

Work by others has shown that high doses of allergens generally producethe best results (i.e., best symptom relief). However, many people areunable to tolerate large doses of allergens because of allergicreactions to the allergens. A peptide can be designed in such a mannerto have the same or enhanced therapeutic properties as the correspondingnaturally-occurring allergen but have reduced side effects (especiallyanaphylactic reactions) can be produced. These can be, for example, apeptide of the present invention (e.g., one having all or a portion ofthe amino acid sequence of Cry j I (SEQ ID NO: 2) or Cry j II (SEQ IDNO: 134)), or a modified peptide, or peptide analogue.

It is also possible to modify the structure of a peptide of theinvention for such purposes as increasing solubility, enhancingtherapeutic or preventive efficacy, or stability (e.g., shelf life exvivo, and resistance to proteolytic degradation in vivo). A modifiedpeptide can be produced in which the amino acid sequence has beenaltered, such as by amino acid substitution, deletion, or addition, tomodify immunogenicity and/or reduce allergenicity, or to which acomponent has been added for the same purpose.

For example, a peptide can be modified so that it maintains the abilityto induce T cell non-responsiveness or reduced T cell responsiveness andbind MHC proteins without to the ability to induce a strongproliferative response or possibly, and proliferative response whenadministered in immunogenic form. In this instance, critical bindingresidues for the T cell receptor can be determined using knowntechniques (e.g., substitution of each residue and determination of thepresence or absence of T cell reactivity). Those residues shown to beessential to interact with the T cell receptor can be modified byreplacing the essential amino acid with another, preferably similaramino acid residue (a conservative substitution) whose presence is shownto enhance, diminish but not eliminate or not affect T cell activity. Inaddition, those amino acid residues which are not essential for T cellreceptor interaction can be modified by being replaced by another aminoacid whose incorporation may enhance, diminish but not eliminate or notaffect T cell activity but does not eliminate binding to relevant MHC.

Additionally, peptides of the invention can be modified by replacing anamino acid shown to be essential to interact with the MHC proteincomplex with another, preferably similar amino acid residue(conservative substitution) whose presence is shown to enhance, diminishbut not eliminate or not affect T cell activity. In addition, amino acidresidues which are not essential for interaction with the MHC proteincomplex but which still bind the MHC protein complex can be modified bybeing replaced by another amino acid whose incorporation may enhance,not affect, or diminish but not eliminate T cell reactivity. Preferredamino acid substitutions for non-essential amino acids include, but arenot limited to substitutions with alanine, glutamic acid, or a methylamino acid.

In order to enhance stability and/or reactivity, peptides of theinvention can also be modified to incorporate one or more polymorphismsin the amino acid sequence of the protein allergen resulting fromnatural allelic variation. Additionally, D-amino acids, non-naturalamino acids or non-amino acid analogues can be substituted or added toproduce a modified protein or peptide within the scope of thisinvention. Furthermore, peptides of the present invention can bemodified using the polyethylene glycol (PEG) method of A. Sehon andco-workers (Wie et al. supra) to produce a protein or peptide conjugatedwith PEG. In addition, PEG can be added during chemical synthesis of aprotein or peptide of the invention. Modifications of proteins orpeptides or portions thereof can also include reduction/ alyklation(Tarr in: Methods of Protein Microcharacterization, J. E. Silvered.Humana Press, Clifton, N.J., pp 155-194 (1986)); acylation (Tarr,supra); chemical coupling to an appropriate carrier (Mishell and Shiigi,eds, Selected Methods in Cellular Immunology, WH Freeman, San Francisco,Calif. (1980); U.S. Pat. No. 4,939,239; or mild formalin treatment(Marsh International Archives of Allergy and Applied Immunology,41:199-215 (1971)).

To facilitate purification and potentially increase solubility ofproteins or peptides of the invention, it is possible to add reportergroup(s) to the peptide backbone. For example, poly-histidine can beadded to a peptide to purify the peptide on immobilized metal ionaffinity chromatography (Hochuli, E. et al., Bio/Technology, 6:1321-1325(1988)). In addition, specific endoprotease cleavage sites can beintroduced, if desired, between a reporter group and amino acidsequences of a peptide to facilitate isolation of peptides free ofirrelevant sequences.

In order to successfully desensitize an individual to a peptide, it maybe necessary to increase the solubility of a peptide for use in bufferedaqueous solutions, such as pharmaceutically acceptable carriers ordiluents, by adding functional groups to the peptide, terminal portionsof the peptide, or by not including hydrophobic T cell epitopes orregions containing hydrophobic epitopes in the peptides or hydrophobicregions of the protein or peptide. For example, to increase solubility,charged amino acids or charged amino acid pairs or triplets may be addedto the carboxy or amino terminus of the peptide. Examples of chargedamino acids include, but are not limited to arginine (R), lysine (K),histidine (H), glutamic acid (E), and aspartic acid (D).

To potentially aid proper antigen processing of T cell epitopes within apeptide, canonical protease sensitive sites can be recombinantly orsynthetically engineered between regions, each comprising at least one Tcell epitope. For example, charged amino acid pairs, such as KK or RR,can be introduced between regions within a peptide during recombinantconstruction of the peptide. The resulting peptide can be renderedsensitive to cathepsin and/or other trypsin-like enzymes cleavage togenerate portions of the peptide containing one or more T cell epitopes.

Site-directed mutagenesis of DNA encoding a peptide or protein of theinvention (e.g. Cry j I or Cry j II or a fragment thereof) can be usedto modify the structure of the peptide or protein by methods known inthe art. Such methods may, among others, include PCR with degenerateoligonucleotides (Ho et al., Gene, 77:51-59 (1989)) or total synthesisof mutated genes (Hostomsky, Z. et al., Biochem. Biophys, Res. Comm.,161:1056-1063 (1989)). To enhance bacterial expression, theaforementioned methods can be used in conjunction with other proceduresto change the eucaryotic codons in DNA constructs encoding protein orpeptides of the invention to ones preferentially used in E. coli, yeast,mammalian cells, or other eukaryotic cells.

Using the structural information now available, it is possible to designCry j I or Cry j II peptides which, when administered to a Japanesecedar pollen sensitive individual in sufficient quantities, will modifythe individual's allergic response to Japanese cedar pollen. This can bedone, for example, by examining the structure of Cry j I or Cry j II,producing peptides (via an expression system, synthetically, chemicalcleavage of the native allergen or otherwise) to be examined for theirability to influence B-cell and/or T-cell responses in Japanese cedarpollen sensitive individuals and selecting appropriate peptides whichcontain epitopes recognized by the cells. In referring to an epitope,the epitope will be the basic element or smallest unit of recognition bya receptor, particularly immunoglobulins, histocompatibility antigensand T cell receptors where the epitope comprises amino acids essentialto receptor recognition. Amino acid sequences which mimic those of theepitopes and which are capable of down regulating allergic response toCry j I or Cry j II can also be used.

It is now also possible to design an agent or a drug capable of blockingor inhibiting the ability of Japanese cedar pollen allergen to induce anallergic reaction in Japanese cedar pollen sensitive individuals. Suchagents could be designed, for example, in such a manner that they wouldbind to relevant anti-Cry j I IgEs, thus preventing IgE-allergen bindingand subsequent mast cell degranulation. Alternatively, such agents couldbind to cellular components of the immune system, resulting insuppression or desensitization of the allergic response to Cryptomeriajaponica pollen allergens.

Peptides of the present invention can also be used for detecting anddiagnosing Japanese cedar pollinosis. For example, this could be done bycombining blood or blood products obtained from an individual to beassessed for sensitivity to Japanese cedar pollen with an isolatedantigenic peptide or peptides of Cry j I, or isolated Cry j I protein,under conditions appropriate for binding of components in the blood(e.g., antibodies, T-cells, B-cells) with the peptide(s) or protein anddetermining the extent to which such binding occurs. Other diagnosticmethods for allergic diseases which the peptides of the presentinvention can be used include radio-allergergosorbent test (RAST), paperradioimmunosorbent test (PRIST), enzyme linked immunosorbent assay(ELISA), radioimmunoassays (RIA), immuno-radiometric assays (IRMA),luminescence immunoassays (LIA), histamine release assays and IgEimmunoblots.

The presence in individuals of IgE specific for at least one proteinallergen and the ability of T cells of the individuals to respond to Tcell epitope(s) of the protein allergen can be determined byadministering to the individuals an Immediate Type Hypersensitivity testand a Delayed Type Hypersensitiity test. The individuals areadministered an Immediate Type Hypersensitivity test (see e.g.Immunology (1985) Roitt, I. M., Brostoff, J., Male, D. K. (eds), C.V.Mosby Co., Gower Medical Publishing, London, N.Y., pp. 19.2-19.18, pp.22.1-22.10) utilizing a peptide of the protein allergen, or a modifiedform of the peptide, each of which binds IgE specific for the allergen.The same individuals are administered a Delayed Type Hypersensitivitytest prior to, simultaneously with, or subsequent to administration ofthe Immediate Type Hypersensitivity test. Of course, if the ImmediateType Hypersensitivity test is administered prior to the Delayed TypeHypersensitivity test, the Delayed Type Hypersensitivity test would begiven to those individuals exhibiting a specific Immediate TypeHypersensitivity reaction. The Delayed Type Hypersensitivity testutilizes a modified form of the protein allergen or a portion thereof,the protein allergen produced recombinantly, or peptide derived from theprotein allergen, each of which has human T cell stimulating activityand each of which does not bind IgE specific for the allergen in asubstantial percentage of the population of individuals sensitive to theallergen (e.g., at least about 75%). Those individuals found to haveboth a specific Immediate Type Hypersensitivity reaction and a specificDelayed Type Hypersensitivity reaction are diagnosed as havingsensitivity to Japanese cedar pollen allergen and may, if need be,administered a therapeutically effective amount of a therapeuticcomposition. The therapeutic composition comprises the modified form ofthe protein or portion thereof, the recombinantly produced proteinallergen, or peptide, each as used in the Delayed Type Hypersensitivitytest, and a pharmaceutically acceptable carrier or diluent.

Peptides comprising at least two regions, each region comprising atleast one T cell epitope of Japanese cedar pollen are also within thescope of the invention. Isolated peptides or regions of isolatedpeptides, each comprising at least two T cell epitopes of a Japanesecedar pollen protein allergen or use of more than one peptide having oneT cell epitope may be desirable for increased therapeutic effectiveness.Peptides which are immunologically related (e.g., by antibody or T cellcross-reactivity) to peptides of the present invention are also withinthe scope of the invention.

Isolated peptides of the invention can be produced as discussedpreviously. With regard to isolated Jun v I or Jun s I peptides,peptides may be produced by biochemically purifying the native Jun v Ior Jun s I proteins from Juniperus virginiana or Juniperus sabinoidespollen as is known in the art, or by recombinant or chemical synthetictechniques as described herein.

To obtain isolated Cry j I or Cry j II peptides of the presentinvention, Cry j I or Cry j II is divided into non-overlapping peptidesof desired length or overlapping peptides of desired lengths which canbe produced recombinantly, or synthetically or by chemical cleavage.Peptides comprising at least one T cell epitope are capable of elicitinga T cell response, such as T cell proliferation or lymphokine secretion,and/or are capable of reducing T cell responsiveness. To determinepeptides comprising at least one T cell epitope, isolated peptides aretested by, for example, T cell biology techniques, to determine whetherthe peptides elicit a T cell response or induce T cellnon-responsivenss. Those peptides found to elicit a T cell response orinduce T cell non-responsiveness are defined as having T cellstimulating activity.

As discussed in Examples 6, 11, and 19 human T cell stimulating activitycan be tested by culturing T cells obtained from an individual sensitiveto Japanese cedar pollen allergen, (i.e., an individual who has an IgEmediated immune response to Japanese cedar pollen allergen) with apeptide derived from the allergen and determining whether proliferationof T cells occurs in response to the peptide as measured, e.g., bycellular uptake of tritiated thymidine. Stimulation indices forresponses by T cells to peptides can be calculated as the maximum countsper minute (CPM) in response to a peptide divided by the control CPM. Astimulation index (S.I.) equal to or greater than two times thebackground level is considered “positive”. Positive results are used tocalculate the mean stimulation index for each peptide for the group ofpatients tested. Preferred peptides of this invention comprise at leastone T cell epitope and have a mean T cell stimulation index of greaterthan or equal to 2.0. A peptide having a mean T cell stimulation indexof greater than or equal to 2.0 is considered useful as a therapeuticagent. Preferred peptides have a mean T cell stimulation index of atleast 2.5, more preferably at least 3.5, more preferably at least 4.0,more preferably at least 5, even more preferably at least 7 and and mostpreferably at least about 9. For example, Cry j I peptides of theinvention having a mean T cell stimulation index of at least 5, as shownin FIG. 14, include CJ1-2 (SEQ ID NO: 27), CJ1-7 (SEQ ID NO: 32), CJ1-10(SEQ ID NO: 35), CJ1-16 (SEQ ID NO: 41), CJ1-17 (SEQ ID NO: 42), CJ1-20(SEQ ID NO: 45), CJ1-22 (SEQ ID NO: 47), CJ1-23 (SEQ ID NO: 48), CJ1-24(SEQ ID NO: 49), CJ1-27 (SEQ ID NO: 52), CJ1-31 (SEQ ID NO: 56), CJ1-32(SEQ ID NO: 57) and CJ1-35 (SEQ ID NO: 60). Peptides of the inventionhaving a mean T cell stimulation index of at least 7, as shown in FIG.14, include CJ1-16 (SEQ ID NO: 41), CJ1-20 (SEQ ID NO: 45, CJ1-22 (SEQID NO: 47), and CJ1-32 (SEQ ID NO: 57).

For therapeutic purposes, preferred peptides are recognized by at least10%, more preferably at least 20%, more preferably at least 30% and evenmore preferably at least 40% or more of individuals in a population ofindividuals sensitive to Japanese cedar pollen. In addition, preferredCry j I peptides have a positivity index (P.I.) of at least about 100,more preferably at least about 250 and most preferably at least about350. The positivity index for a peptide is determined by multiplying themean T cell stimulation index by the percent of individuals, in apopulation of individuals sensitive to Japanese cedar pollen (e.g.,preferably at least 15 individuals, more preferably at least 30individuals or more), who have a T cell stimulation index to suchpeptide of at least 2.0. Thus, the positivity index represents both thestrength of a T cell response to a peptide (S.I.) and the frequency of aT cell response to a peptide in a population of individuals sensitive toJapanese cedar pollen. For example, as shown in FIG. 14, peptide CJ1-22(SEQ ID NO: 47) has a mean S.I. of 14.5 and 60.0% of positive responsesin the group of individuals tested resulting in a positivity index of870.00. Peptides of Cry j I having a positivity index of at least about100 and a mean T cell stimulation index of at least about 4 include:CJ1-16 (SEQ ID NO: 41), CJ1-17 (SEQ ID NO: 42), CJ1-20 (SEQ ID NO: 45),CJ1-22 (SEQ ID NO: 47), CJ1-23 (SEQ ID NO: 48), CJ1-24 (SEQ ID NO: 49),CJ1-26 (SEQ ID NO: 51), CJ1-27 (SEQ ID NO: 52), CJ1-32 (SEQ ID NO: 57)and CJ1-35 (SEQ ID NO: 60).

In order to determine precise T cell epitopes by, for example, finemapping techniques, a peptide having T cell stimulating activity andthus comprising at least one T cell epitope as determined by T cellbiology techniques is modified by addition or deletion of amino acidresidues at either the amino or carboxy terminus of the peptide andtested to determine a change in T cell reactivity to the modifiedpeptide. If two or more peptides which share an area of overlap in thenative protein sequence are found to have human T cell stimulatingactivity, as determined by T cell biology techniques, additionalpeptides can be produced comprising all or a portion of such peptidesand these additional peptides can be tested by a similar procedure.Following this technique, peptides are selected and producedrecombinantly or synthetically. Example 11 discusses preferred peptidesof the invention produced in accordance with these techniques.

For therapeutic purposes, peptides are selected based on variousfactors, including the strength of the T cell response to the peptide(e.g., stimulation index), the frequency of the T cell response to thepeptide in a population of individuals sensitive to Japanese cedarpollen, and the potential cross-reactivity of the peptide with otherallergens from other species of trees as discussed earlier (e.g.Cupressus sempervirens, Cupressus arizonica, Juniperus virginiana,Juniperus sabinoides, Chamae cyparisobtusa, etc.) or ragweed (Amb a1.1). The physical and chemical properties of these selected peptides(e.g., solubility, stability) are examined to determine whether thepeptides are suitable for use in therapeutic compositions or whether thepeptides require modification as described herein.

To determine whether a peptide (candidate peptide) or a combination ofcandidate peptides are likely to comprise a sufficient percentage of theT cell epitopes of the protein antigen of interest to induce T cellnonresponsiveness in a substantial percentage of a population ofindividuals sensitive to the protein antigen, an algorithm can be used.In accordance with one such algorithm, a set of overlapping peptides isproduced by systematically dividing the protein sequence of the allergenor other antigen into at least two overlapping peptide regions ofdesired lengths (e.g., of about 12-30 amino acid residues in length,preferably not longer than about 25 amino acid residues in length withabout 5-15 amino acid residues of overlap). This division into peptideregions can be arbitrary, can be made according to an algorithm, or canbe wholly or partially based on regions of the protein antigen known tocomprise at least one T cell epitope. Preferably, at least 50% of theentire protein sequence of the protein allergen or other protein antigenand more preferably, the entire protein sequence of the protein allergenor other protein antigen and more preferably, the entire proteinsequence of the protein allergen or other protein antigen is dividedinto two or more peptides. A human T cell stimulation index isdetermined for each of the peptides in an in vitro T cell proliferationassay as described herein for each individual tested in a population ofindividuals sensitive to the protein antigen. A candidate peptide orcombination of candidate peptides is selected based, at least in part,on the mean human T cell stimulation index of the candidate peptide inthe set of peptides tested and the positivity index of the candidatepeptide in the set of peptides tested. The human T cell stimulationindex for the candidate peptide(s) is summed. For each individual, thehuman T cell stimulation index for the candidate peptide(s) is dividedby the sum of the human T cells stimulation indices of the remainingpeptides in the set of peptides tested to determine a percent of T cellreactivity as shown below:

$\begin{matrix}{{\%\mspace{14mu} T\mspace{14mu}{Cell}\mspace{14mu}{Reactivity}\mspace{14mu}{of}\mspace{14mu} a\mspace{14mu}{candidate}\mspace{14mu}{{peptide}(s)}} = {\frac{{Candidate}\mspace{11mu}{S.I.}}{{Sum}\mspace{14mu}{of}\mspace{14mu}{S.I.\mspace{14mu}{of}}\mspace{14mu}{the}\mspace{14mu}{set}\mspace{14mu}{of}\mspace{14mu}{Overlapping}\mspace{14mu}{peptides}} \times 100}} & (1)\end{matrix}$

Alternatively, the presence of T cell epitopes in the candidate peptidedependent on amino acids residues in an overlapping peptide located ateither the N-terminus or C-terminus of the candidate peptide in theamino acid sequence of the protein antigen, but which epitopes are notpresent in the candidate peptide can be considered in calculating thepercent of T cell reactivity in the candidate peptide by use of thefollowing formula:

$\begin{matrix}{{\%\mspace{14mu} T\mspace{14mu}{Cell}\mspace{14mu}{Reactivity}\mspace{14mu}{of}\mspace{14mu} a\mspace{14mu}{candidate}\mspace{14mu}{{peptide}(s)}} = {\frac{N_{T}\mspace{11mu}{flanking}\mspace{14mu}{peptide}\mspace{14mu}{S.I.{+ {Candidate}}}\mspace{14mu}{peptide}\mspace{14mu}{S.I.{+ C_{T}}}\mspace{11mu}{flanking}\mspace{14mu}{peptide}\mspace{14mu}{S.I.}}{{Sum}\mspace{14mu}{of}\mspace{14mu}{S.I.\mspace{14mu}{of}}\mspace{14mu}{the}\mspace{14mu}{set}\mspace{14mu}{of}\mspace{14mu}{overlapping}\mspace{14mu}{peptides}} \times 100}} & (2)\end{matrix}$

In this formula, “N_(T) flanking peptide” refers to a peptide whichcomprises amino acid residues which overlap with amino acid residueslocated at the N-terminus of the candidate peptide in the amino acidsequence of the protein antigen from which the peptide is derived;“C_(T) flanking peptide” refers to a peptide which comprises amino acidresidues which overlap with amino acid residues located a the C-terminusof the candidate peptide in the amino acid sequence of the proteinantigen from which the peptide is derived. In this calculationstimulation indices for the candidate peptide, the N-terminal flankingpeptide and the C-terminal flanking peptide are added and divided by thesum total of the stimulation indices for the entire set of overlappingpeptides obtain a percent of T cell reactivity for the candidatepeptide. If a combination of two or more candidate peptides is selectedeach of which contains amino acid residues which overlap, thiscalculation cannot be used to determine a percent of T celI reactivityfor each candidate peptide separately. However, a total percent of Tcell reactivity for the combination of candidate peptides can beobtained. In this situation, the stimulation indices for all of thecandidate peptides which overlap is included in the calculation.

The values obtained for the percentage of T cell reactivity for thecandidate peptide or combination of peptides in each individual testedcan be expressed as a range of the lower and higher values of theresults of the above described calculations. By either of the abovecalculations, the percent is obtained for at least about twenty (20) andpreferably at least about thirty (30) individuals sensitive to theprotein antigen and a mean percent is determined. For use in thecompositions of the invention, the candidate peptide or combination ofcandidate peptides has the following criteria: (I) the candidate peptideor combination of candidate peptides has a mean percent of at leastabout 10%, preferably at least about 20%, more preferably at least about30%, more preferably at least about 40% and more preferably at leastabout 50-60% or greater; and (2) in the population of individuals testedat least about 60%, preferably at least about 75%, and more preferablyat least about 90-100% have positive T call responses (S.I. equal to orgreater than 2.0) in response to the candidate peptide or combination ofcandidate peptides. A candidate peptide or combination of candidatepeptides meeting the above criteria is likely to comprise a sufficientpercentage of the T cell epitopes of the protein antigen to induce Tcell nonresponsiveness in a substantial percentage of a population ofindividuals sensitive to the protein antigen.

As an illustrative embodiment of the above-described algorithm, a set ofoverlapping peptides derived from Cry j I were produced and tested.Secondary T cell cultures determined to be reactive with Cry j I proteinantigen were derived from 36 Cry j I-allergic subjects and analyzed forreactivity to the overlapping set of peptides in an in vitro T cellproliferation assay as described herein. The results are shown in FIG.45. The highest stimulation index greater than or equal to 2.0 inresponse to each peptide was recorded for each subject tested. The datawere then analyzed by the equations above. The results and calculationsof the percent of T cell reactivity for a single Cry j I-allergicsubject are shown below using formulas (1) and (2).

T CELL REACTIVITY FOR PATIENT 1308 PEPTIDE STIMULATION INDEX CJ1-1 10.9CJ1-2 16.1 CJ1-3 8.8 CJ1-4 0 CJ1-5 3.2 CJ1-6 0 CJ1-7 2.5 CJ1-8 0 CJ1-418.9 CJ1-11 0 CJ1-12 0 CJ1-13 0 CJ1-14 0 CJ1-15 0 CJ1-42.5 17.6 CJ1-18 0CJ1-19 0 CJ1-20 0 CJ1-21 0 CJ1-43.39 25.6 CJ1-23 5.3 CJ1-24.5 6.9 CJ1-259.4 CJ1-26 11.9 CJ1-27 5.5 CJ1-28 0 CJ1-29 2.9 CJ1-30 0 CJ1-44.8 21.5CJ1-33 20.9 CJ1-34 17.8 CJ1-35 0 SUM OF STIMULATION INDICES: 195.7(DENOMINATOR)

% Reactivity of Peptides 44.8 for patient 1308

$\begin{matrix}{\frac{{{CJ}\; 1} - {44.8\left( {S.I.} \right)}}{195.7} = {{\frac{21.5}{195.7} \times 100} = {11\%}}} & (1) \\{\frac{{{CJ}\; 1} - 30 + {{CJ}\; 1} - 44.8 + {{CJ}\; 1} - {33\left( {S.I.s} \right)}}{195.7} = {{\frac{0 + 21.5 + 20.9}{195.7} \times 100} = {21.7\%}}} & (2)\end{matrix}$

Therefore the estimated range of T cell reactivity for Peptide 44.8 forthis patient is 11%-21.7% of the total reactivity of the Cry j Iprotein. The above calculation is repeated for any potential candidatepeptides. In the population of 36 Cry j I-allergic subjects tested thefollowing results were obtained:

Range Frequency of mean of response percentage at least Candidate T Cellone Peptides Reactivity peptide CJ1-24.5 + 36-53% 97% CJ1-43.3 +CJ1-44.8

Additionally, for therapeutic purposes, preferred T cellepitope-containing peptides of the invention do not bind immunoglobulinE (IgE) or bind IgE to a substantially lesser extent (i.e., preferablyat least 100-fold less or more preferably at least 1,000-fold less) thanthe protein allergen from which the peptide is derived binds IgE. Themajor complications of standard immunotherapy are IgE-mediated responsessuch as anaphylaxis. Immunoglobulin E is a mediator of anaphylacticreactions which result from the binding and cross-linking of antigen toIgE on mast cells or basophils and the release of mediators (e.g.,histamine, serotonin, eosinophil chemotacic factors). Thus, anaphylaxisin a substantial percentage of a population of individuals sensitive toCry j I or Cry j II could be avoided by the use in immunotherapy of apeptide or peptides which do not bind IgE in a substantial percentage(e.g., at least about 75%) of a population of individuals sensitive toCry j I allergen, or if the peptide binds IgE, such binding does notresult in the release of mediators from mast cells or basophils. Therisk of anaphylaxis could be reduced by the use in immunotherapy of apeptide or peptides which have reduced IgE binding. Moreover, peptideswhich have minimal IgE stimulating activity are desirable fortherapeutic effectiveness. A T cell epitope-containing peptide of theinvention, when administered to a Japanese cedar pollen-sensitiveindividual, is capable of modifying the allergic response of theindividual to the allergen.

A preferred isolated peptide of the invention comprises at least one Tcell epitope of the Japanese cedar pollen allergen, Cry j I or Cry j IIand accordingly the peptide comprises at least approximately seven aminoacid residues. For purposes of therapeutic effectiveness, therapeuticcompositions of the invention may comprise peptides having at least twoT cell epitopes of Cry j I or Cry j II, and accordingly, the peptidecomprises at least approximately eight amino acid residues andpreferably at least fifteen amino acid residues. Alternatively, theindividual sensitive to Cry j I or Cry j II may be administered morethan one peptide of the invention comprising at least one T cellepitope. Additionally, therapeutic compositions comprising preferredisolated peptides of the invention preferably comprise a sufficientpercentage of the T cell epitopes of the entire protein allergen suchthat a therapeutic regimen of administration of the composition to anindividual sensitive to Japanese cedar pollen, results in T cells of theindividual being renedered non-responsive to the protein allergen.Peptides of the invention produced by chemical synthesis comprising upto approximately forty-five amino acid residues in length, and mostpreferably up to approximately thirty amino acid residues in length areparticularly desirable as increases in length may result in difficultyin peptide synthesis. Peptides of the invention may also be producedrecombinantly as described above or by chemical cleavage of the nativeallergen.

Preferred peptides comprise all or a portion of the areas of major Tcell reactivity within Cry j I or Cry j II. Areas of major T cellreactivity within Cry j I are designated herein as, Region 1, Region 2,Region 3, Region 4 and Region 5. Each major area of T cell activity isdefined as follows and is shown in FIG. 4 a-b. Region 1 comprises aminoacid residues 1-50 of Cry j I (SEQ ID NO: 61); Region 2 comprises aminoacid residues 61-120 of Cry j I (SEQ ID NO: 62); Region 3 comprisesamino acid residues 131-180 of Cry j I (SEQ ID NO: 63); Region 4comprises amino acid residues 191-280 of Cry j I (SEQ ID NO: 64); Region5 comprises amino acid residues 291-353 of the Cry j I (SEQ ID NO: 65).Preferred areas of major T cell reactivity within each Region as shownin FIG. 4 a-b and comprise: amino acid residues 1-40 (SEQ ID NO: 66);amino acid residues 81-110 (SEQ ID NO: 67); amino acid residues 151-180(SEQ ID NO: 68); amino acid residues 191-260 (SEQ ID NO: 69); and aminoacid residues 291-330 (SEQ ID NO: 70).

Peptides derived from the Cry j I protein allergen which can be used fortherapeutic purposes comprise all or a portion of the followingpeptides: CJ1-1 (SEQ ID NO: 26). CJ1-2 (SEQ ID NO: 27), CJ1-3 (SEQ IDNO: 28), CJ1-4 (SEQ ID NO: 29), CJ1-7 (SEQ ID NO: 32), CJ1-8 (SEQ ID NO:33), CJ1-9 (SEQ ID NO: 34), CJ1-10 (SEQ ID NO: 35), CJ1-11 (SEQ IDNO:36), CJ1-12 (SEQ ID NO:37), CJ1-14 (SEQ ID NO: 39), CJ1-15 (SEQ IDNO: 40), CJ1-16 (SEQ ID NO: 41), CJ1-17 (SEQ ID NO: 42), CJ1-18 (SEQ IDNO: 43), CJ1-19 (SEQ ID NO: 44), CJ1-20 (SEQ ID NO: 45), CJ1-21 (SEQ IDNO: 46), CJ1-22 (SEQ ID NO: 47), CJ1-23 (SEQ ID NO: 48), CJ1-24 (SEQ IDNO: 49), CJ1-25 (SEQ ID NO: 50), CJ1-26 (SEQ ID NO: 51), CJ1-27 (SEQ IDNO: 52), CJ1-28 (SEQ ID NO: 53), CJ1-30 (SEQ ID NO: 55), CJ1-31 (SEQ IDNO: 56), CJ1-32 (SEQ ID NO: 57), CJ1-33 (SEQ ID NO: 58), CJ1-34 (SEQ IDNO: 59) and CJ1-35 (SEQ ID NO: 60) wherein the portion of the peptidepreferably has a mean T cell stimulation index equivalent to, or greaterthan the mean T cell stimulation index of the peptide from which it isderived as shown in FIG. 14.

Preferably peptides derived from the Cry j I protein allergen which canbe used for therapeutic purposes comprise all or a portion of thefollowing peptides: CJ1-2 (SEQ ID NO: 27), CJ1-9 (SEQ ID NO: 34), CJ1-10(SEQ ID NO: 35), CJ1-16 (SEQ ID NO: 41), CJ1-17 (SEQ ID NO: 42), CJ1-20(SEQ ID NO: 45), CJ1-22 (SEQ ID NO: 47), CJ1-23 (SEQ ID NO: 48), CJ1-24(SEQ ID NO: 49), CJ1-25 (SEQ ID NO: 50), CJ1-26 (SEQ ID NO: 51), CJ1-27(SEQ ID NO: 52), CJ1-30 (SEQ ID NO: 53), CJ1-31 (SEQ ID NO: 54), CJ1-32(SEQ ID NO: 56)and CJ1-35 (SEQ ID NO: 60) wherein the portion of thepeptide preferably has a mean T cell stimulation index equivalent to, orgreater than the mean T cell stimulation index of the peptide from whichit is derived as shown in FIG. 14.

Additionally, other peptides useful for therapeutic purposes comprisethe following peptides: CJ1-41 (SEQ ID NO: 71), CJ1-41.1 (SEQ ID NO:72), CJ1-41.2 (SEQ ID NO: 73), CJ1-41.3 (SEQ ID NO: 74), CJ1-42 (SEQ IDNO: 75), CJ1-42.1 (SEQ ID NO: 76), CJ1-42.2 (SEQ ID NO: 77), CJ1-43 (SEQID NO: 78), CJ1-43.1 (SEQ ID NO: 79), CJ1-43.6 (SEQ ID NO: 80), CJ1-43.7(SEQ ID NO: 81), CJ1-43.8 (SEQ ID NO: 82), CJ1-43.9 (SEQ ID NO: 83),CJ1-43.10 (SEQ DD NO: 84), CJ1-43.11 (SEQ ID NO: 85), CJ1-43.12 (SEQ IDNO: 86), CJ1-45 (SEQ ID NO: 87), CJ1-45.1 (SEQ ID NO: 88), CJ1-45.2 (SEQID NO: 89), CJ1-44 (SEQ ID NO: 90), CJ1-44.1 (SEQ ID NO: 91), CJ1-44.2(SEQ ID NO: 92) and CJ1-44.3 (SEQ ID NO: 93), all as shown in FIG. 18.Some of these peptides have been further modified for the purpose ofincreasing their solubility Such modified peptides derived from Cry j Icomprise all or a portion of the following peptides: CJ1-42.3, CJ1-42.4,CJ1-42.5 (SEQ ID NO: 119), CJ1-42.6, CJ1-42.7, CJ1-42.8 (SEQ ID NO:120), CJ1-42.9, CJ1-42.10, CJ1-42.1 1, CJ1-42.12, CJ1-42.13, CJ1-42.14,42.15, CJ1-43.2, CJ1-43.3, CJ1-43.4, 43.5, CJ1-43.12, CJ1-43.13,CJ1-43.14, CJ1-43.15, CJ1-43.16, CJ1-43.17, CJ1-43.18, CJ1-43.19,CJ1-43.20, CJ1-43.21, CJ1-43.22, CJ1-43.23, CJ1-43.24, CJ1-43.26,CJ1-43.26 (SEQ ID NO: 12 1), CJ1-43.27 (SEQ ID NO: 122), CJ1-43.28,CJ1-43.29, CJ1-43.30 (SEQ ID NO: 123), CJ1-43.31 (SEQ ID NO: 124),CJ1-43.32 (SEQ ID NO: 125), CJ1-43.33, CJ1-43.34, CJ1-43.35 (SEQ ID NO:126), CJ1-43.36 (SEQ ID NO: 127), CJ1-43.37, CJ1-43.38, CJ1-43.39 (SEQID NO: 128), CJ1-43.40, CJ1-43.41, CJ1-43.42, CJ1-43.43, CJ1-43.44,CJ1-43.45, C1-43.46, CJ1-43.47, CJ1-43.48, CJ1-43.49, CJ1-43.50,CJ1-43.51, CJ1-43.52, CJ1-43.53, CJ1-43.54, CJ1-43.55, CJ1-43.56,CJ1-43.57, CJ1-43.58, CJ1-43.59, CJ1-43.60, CJ1-24.2, CJ1-24.5 (SEQ IDNO: 129), CJ1-44.5 (SEQ ID NO: 130), CJ1-44.6 (SEQ ID NO: 131),CJ1-44.7, CJ1-44.8 (SEQ ID NO: 132), CJ1-44.9, CJ1-44.10 all as shown inFIG. 18, 20, or 44). Preferred peptides which have been modified forenhanced solubility include the following peptides: CJ1-42.5 (SEQ ID NO:119), CJ1-42.8 (SEQ ID NO: 120), CJ1-43.26 (SEQ ID NO: 121), CJ1-43.27(SEQ ID NO: 122), CJ1-43.30 (SEQ ID NO: 123), CJ1-43.31 (SEQ ID NO:124), CJ1-43.32 (SEQ ID NO: 125), CJ1-43.35 (SEQ ID NO: 126), CJ1-43.36(SEQ ID NO: 127), CJ1-43.39 (SEQ ID NO: 128), CJ1-24.5 (SEQ ID NO: 129),CJ1-44.5 (SEQ ID NO: 130), CJ1-44.6 (SEQ ID NO: 131), CJ1-44.8 (SEQ IDNO: 132) and CJ1-44.9, all as shown in FIGS. 20 and 44.

Of the above group of modified peptides, several peptides have beenidentified as “unique” modified peptides. A “unique” modified peptide isdefined herein as a modified peptide which 1) possesses thecharacteristic of “superior solubility”; 2) has T cell reactivity whichis similar to that of the “parent” peptide from which the “unique”modified peptide is derived; and 3) is stable in an aqueous buffer at apH ranging from pH6 to pH8. “Superior solubility” is defined herein assolubility of greater than 5 mg/ml over a pH range of pH6 to pH8 in anaqueous buffer. Certain modified peptides are characterized as “unique”due to the difficulties encountered when developing a modified peptidewhich meets all of the stringent requirements of a “unique” peptidedefined herein. In many cases, multiple modifications of a parentpeptide are attempted prior to identifying a modified derivitive peptidewhich meets all the characteristics of a “unique” modified peptide.Unique modified peptides are particularly useful as candidate peptidesfor formulating injectable multipeptide therapeutic formulations of theinvention because “unique” modified peptides are soluble and stable inthe same physiologically acceptable pH range as well as elicit thenecessary T cell reactivity of a therapeutic peptide of the invention.“Unique” modified peptides of the invention include but are not limitedto the following group of modified peptides: CJ1-24.5, CJ1-43.39,CJ1-43.50, CJ1-44.8, and CJ1-44.9 all as shown in FIGS. 20 and 44.Example 21 describes the development and identification of “unique”modified peptides of the invention.

Preferred peptides of Cry j II which may comprise T cell epitopesinclude: Cry j IIA (SEQ ID NO: 185) Cry j IIB (SEQ ID NO: 186) and Cry jIIQ (SEQ ID NO: 193) (FIG. 41). Preferred Cry j II peptides comprising Tcell epitopes include: Cry j IIC, Cry j IID, Cry j IIE, (SEQ ID NO: 189)Cry j IIF (SEQ ID NO: 190), Cry j IIG (SEQ ID NO: 191) and Cry j IIH(SEQ ID NO: 192) all as shown in FIG. 41.

One embodiment of the present invention features a peptide or portionthereof of Cry j I which comprises at least one T cell epitope of theprotein allergen and has a formula X_(n)-Y-Z_(m). According to theformula, Y is an amino acid sequence selected from the group of Cry j Ipeptides consisting of CJI-1 (SEQ ID NO: 26), CJI-2 (SEQ ID NO: 27),CJI-3 (SEQ ID NO: 28), CJI-4 (SEQ ID NO: 29), CJI-7 (SEQ ID NO: 32),CJI-8 (SEQ ID NO: 33), CJI-9 (SEQ ID NO: 34), CJI-10 (SEQ ID NO: 35),CJI-11 (SEQ ID NO: 36), CJI-12 (SEQ ID NO: 37), CJI-14 (SEQ ID NO: 39),CJI-15 (SEQ ID NO: 40), CJI-16 (SEQ ID NO: 41), CJI-17 (SEQ ID NO: 42),CJI-18 (SEQ ID NO: 43), CJI-19 (SEQ ID NO: 44), CJI-20 (SEQ ID NO: 45),CJI-21 (SEQ ID NO: 46), CJI-22 (SEQ ID NO: 47), CJI-23 (SEQ ID NO: 48),CJI-24 (SEQ ID NO: 49), CJI-25 (SEQ ID NO: 50), CJI-26 (SEQ ID NO: 51),CJI-27 (SEQ ID NO: 52), CJI-28 (SEQ ID NO: 53), CJI-30 (SEQ ID NO: 55),CJI-31 (SEQ ID NO: 56), CJI-32 (SEQ ID NO: 57), CJI-33 (SEQ ID NO: 58),CJI-34 (SEQ ID NO: 59), CJI-35 (SEQ ID NO: 60), CJI-41, CJI-42.5 (SEQ IDNO: 119), CJI-42.8 (SEQ ID NO: 120), CJI-43.26 (SEQ ID NO: 121),CJI-43.27 (SEQ ID NO: 122), CJI-43.30 (SEQ ID NO: 123), CJI-43.31 (SEQID NO: 124), CJI-43.32 (SEQ ID NO: 125), CJI-43.35 (SEQ ID NO: 126),CJI-43.36 (SEQ ID NO: 127), CJI-43.39 (SEQ ID NO: 128), CJI-24.5 (SEQ IDNO: 129), CJI-44.5 (SEQ ID NO: 130), CJI-44.6 (SEQ ID NO: 131), CJI-44.8(SEQ ID NO: 132) and preferably selected from the group consisting ofCJI-2 (SEQ ID NO: 27), CJI-9 (SEQ ID NO: 29), CJI-10 (SEQ ID NO: 30),CJI-16 (SEQ ID NO: 41), CJI-17 (SEQ ID NO: 42), CJI-20 (SEQ ID NO: 45),CJI-22 (SEQ ID NO: 47), CJI-23 (SEQ ID NO: 48), CJI-24 (SEQ ID NO: 49),CJI-25 (SEQ ID NO: 50), CJI-26 (SEQ ID NO: 51), CJI-27 (SEQ ID NO: 52),CJI-30 (SEQ ID NO: 55), CJI-31 (SEQ ID NO: 56), CJI-32 (SEQ ID NO: 57),CJI-35 (SEQ ID NO: 60) CJI-41, CJI-24,5 (SEQ ID NO: 129), CJI-43.39 (SEQID NO: 128) and CJI-44.8 (SEQ ID NO: 132). In addition, X_(n) are aminoacid residues contiguous to the amino terminus of Y in the amino acidsequence of the protein allergen and Z_(m) are amino acid residuescontiguous to the carboxy terminus of Y in the amino acid sequence ofthe protein allergen. Preferably, the amino acids comprising the aminoterminus of X and the carboxy terminus of Z are selected from chargedamino acids, i.e., arginine (R), lysine (K), histidine (H), glutamicacid (E) or aspartic acid (D); amino acids with reactive side chains,e.g., cysteine (C), asparagine (N) or glutamine (Q); or amino acids withsterically small side chains, e.g. alanine (A) or glycine (G). In theformula, n is preferably 0-30 and m is preferably 0-30. Preferably n andm are 0-5, and most preferably n+m is less than 10. Preferably, thepeptide or portion thereof has a mean T cell stimulation indexequivalent to or greater than the mean T cell stimulation index of Y asshown in FIG. 14. Y may also be selected from the group of Cry j IIpeptides consisting of Cry j IIA (SEQ ID NO: 185), Cry j IIB (SEQ ID NO:186), Cry j IIC (SEQ ID NO: 187), Cry j IID (SEQ ID NO: 188), Cry j IIE(SEQ ID NO: 189), Cry j IIF (SEQ ID NO: 190), Cry j IIG (SEQ ID NO:191), Cry j IIH (SEQ ID NO: 192), or Cry j IIQ (SEQ ID NO: 193) all asshown in FIG. 41.

Another embodiment of the present invention provides peptides comprisingat least two regions, each region comprising at least one T cell epitopeof Cry j I or Cry j II and accordingly each region comprises at leastapproximately seven amino acid residues. These peptides comprising atleast two regions can comprise as many amino acid residues as desiredand preferably comprise 14 amino acid residues of a Cry j I or Cry j IIallergen, or even more preferably about 30 amino acid residues and mostpreferably at least about 40 amino acid residues of Cry j I or Cry j IIallergen. If desired, the amino acid sequences of the regions can beproduced and joined by a linker to increase sensitivity to processing byantigen-presenting cells. Such linker can be any non-epitope amino acidsequence or other appropriate linking or joining agent. To obtainpreferred peptides comprising at least two regions, each comprising atleast one T cell epitope, the regions are arranged in a configurationdifferent from a naturally-occurring configuration of the regions in theallergen. For example, the regions containing T cell epitope(s) can bearranged in a noncontiguous configuration and can preferably be derivedfrom the same protein allergen. Noncontiguous is defined as anarrangement of regions containing T cell epitope(s) which is differentthan that of an amino acid sequence present in the protein allergen fromwhich the regions are derived. Furthermore, the noncontiguous regionscontaining T cell epitopes can be arranged in a nonsequential order(e.g., in an order different from the order of the amino acids of thenative protein allergen from which the region containing T cellepitope(s) are derived in which amino acids are arranged from an aminoterminus to a carboxy terminus). A peptide for use as a therapeutic cancomprise at least 15%, at least 30%, at least 50% or up to 100% of the Tcell epitopes of Cry j I or Cry j II but does not comprise the wholeprotein sequence of the allergen.

The individual peptide regions can be produced and tested to determinewhich regions bind immunoglobulin E specific for Cry j I and which ofsuch regions would cause the release of mediators (e.g., histamine) frommast cells or basophils. Those peptide regions found to bindimmunoglobulin E and cause the release of mediators from mast cells orbasophils in greater than approximately 10-15% of the allergic seratested are preferably not included in the peptide regions arranged toform preferred peptides of the invention.

Additionally, regions of a peptide of the invention preferably compriseall or a portion of the above discussed preferred areas of major T cellreactivity within Cry j II or Cry j I (i.e., Regions 1-5 of Cry j I) orthe above discussed preferred areas of major T cell activity within eachRegion (i.e. amino acids from residues 1-40, 81-110, 151-180, 191-260and 291-330 of Cry j (SEQ ID NO: 2)). For example, with regard to Cry jI, one region can comprise all or a portion of Region I (amino acidresidues 1-51) (SEQ ID NO: 61)and one region can comprise all or aportion of Region 2 (amino acid residues 61-120). (SEQ ID NO: 62)Peptides of the invention can comprise all or a portion of two or moreof these Regions (i.e., Regions 1-5) and preferred resulting peptides donot bind IgE and cause the release of mediators from most cells orbasophils. Preferred peptides derived from Cry j I comprise all or aportion of Region 3 (SEQ ID NO: 63), Region 4 (SEQ ID NO: 64) and Region5 (SEQ ID NO: 65), and, optionally, Region 1 (SEQ ID NO: 61)or Region2.(SEQ ID NO: 62) Further, if one of these Regions is found to bind IgEand cause the release of mediators from mast cells or basophils, then itis preferred that the peptide not comprise such Region, but rathercomprises various regions derived from such Region which do not bind IgEor cause release of mediators from mast cells or basophils.

Examples of preferred regions of Cry j I include: CJ1-1 (SEQ ID NO: 26),CJ1-2 (SEQ ID NO: 27), CJ1-3 (SEQ ID NO: 28), CJ1-4 (SEQ ID NO: 29),CJ1-7 (SEQ ID NO: 32), CJ1-8 (SEQ ID NO: 33), CJ1-9 (SEQ ID NO: 34),CJ1-10 (SEQ ID NO: 35), CJ1-11 (SEQ ID NO: 36), CJ1-12 (SEQ ID NO: 37),CJ1-14 (SEQ ID NO: 39), CJ1-15 (SEQ ID NO: 40), CJ1-16 (SEQ ID NO: 41),CJ1-17 (SEQ ID NO: 42), CJ1-18 (SEQ ID NO: 43), CJ1-19 (SEQ ID NO: 44),CJ1-20 (SEQ ID NO: 45), CJ1-21 (SEQ ID NO: 46), CJ1-22 (SEQ ID NO: 47),CJ1-23 (SEQ ID NO: 48), CJ1-24 (SEQ ID NO: 49), CJ1-25 (SEQ ID NO: 50),CJ1-26 (SEQ ID NO: 51), CJ1-27 (SEQ ID NO: 52), CJ1-28 (SEQ ID NO: 53),CJ1-30 (SEQ ID NO: 55), CJ1-31 (SEQ ID NO: 56), CJ1-32 (SEQ ID NO: 57),CJ1-33 (SEQ ID NO: 58), CJ1-34 (SEQ ID NO: 59), CJ1-35 (SEQ ID NO: 60),CJ1-42.5 (SEQ ID NO: 119), CJ1-42.8 (SEQ ID NO: 120), CJ1-43.26 (SEQ IDNO: 121), CJ1-43.27, (SEQ ID NO: 122) CJ1-43.30 (SEQ ID NO: 123),CJ1-43.31 (SEQ ID NO: 124), CJ1-43.32 (SEQ ID NO: 125), CJ1-43.35 (SEQID NO: 126), CJ1-43.36 (SEQ ID NO: 127), CJ1-43.39 (SEQ ID NO: 128),CJ1-24.5 (SEQ ID NO: 129), CJ1-44.5 (SEQ ID NO: 130), CJ1-44.6 (SEQ IDNO: 131), CJ1-44.8 (SEQ ID NO: 132), the amino acid sequences of suchregions being shown in FIG. 13 and FIG. 20, or portions of said regionscomprising at least one T cell epitope.

Preferred peptides comprise various combinations of two or more regions,each region comprising all or a portion of the above-discussed preferredareas of major T cell reactivity. Preferred peptides comprise acombination of two or more regions (each region having an amino acidsequence as shown in FIG. 13 and FIG. 20), including:

CJ1-1, (SEQ ID NO: 26) CJ1-2 (SEQ ID NO: 27) and CJ1-3; (SEQ ID NO: 28)CJ1-1 (SEQ ID NO: 26) and CJ1-2; (SEQ ID NO: 27) CJ1-9 (SEQ ID NO: 34)and CJ1-10; (SEQ ID NO: 35) CJ1-14, (SEQ ID NO: 39) CJ1-15, (SEQ ID NO:40) CJ1-16 (SEQ ID NO: 41) and CJ1-17; (SEQ ID NO: 42) CJ1-20, (SEQ IDNO: 45) CJ1-21, (SEQ ID NO: 46) CJ1-22, (SEQ ID NO: 47) CJ1-23; (SEQ IDNO: 48) CJ1-20, (SEQ ID NO: 45) CJ1-22 (SEQ ID NO: 47) and CJ1-23; (SEQID NO: 48) CJ1-22 (SEQ ID NO: 47) and CJ1-23; (SEQ ID NO: 48) CJ1-22,(SEQ ID NO: 47) CJ1-23 (SEQ ID NO: 48) and CJ1-24; (SEQ ID NO: 49)CJ1-24 (SEQ ID NO: 49) and CJ1-25; (SEQ ID NO: 50) CJ1-30, (SEQ ID NO:55) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-31 (SEQ IDNO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-22, CJ1-23, (SEQ ID NO: 48)CJ1-16 (SEQ ID NO: 41) and CJ1-17; (SEQ ID NO: 42) CJ1-22, (SEQ ID NO:47) CJ1-23, (SEQ ID NO: 48) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ IDNO: 57) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-31 (SEQ IDNO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-9, (SEQ ID NO: 34) CJ1-10 (SEQID NO: 35) and CJ1-16; (SEQ ID NO: 41) CJ1-16 (SEQ ID NO: 41) andCJ1-17; (SEQ ID NO: 42) CJ1-17, (SEQ ID NO: 42) CJ1-22 (SEQ ID NO: 47)and CJ1-23; (SEQ ID NO: 48) CJ1-16, (SEQ ID NO: 41) CJ1-17 (SEQ ID NO:42) and CJ1-20; (SEQ ID NO: 45) CJ1-31, (SEQ ID NO: 56) CJ1-32 (SEQ IDNO: 57) and CJ1-20; (SEQ ID NO: 45) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQID NO: 48) CJ1-1, (SEQ ID NO: 26) CJ1-2 (SEQ ID NO: 27) and CJ1-3; (SEQID NO: 28) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-22 (SEQID NO: 47) and CJ1-23, (SEQ ID NO: 48) CJ1-31 (SEQ ID NO: 56) andCJ1-32; (SEQ ID NO: 57) CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQ ID NO: 35)CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-22 (SEQ ID NO: 47)and CJ1-23; (SEQ ID NO: 48) CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQ ID NO:35) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-31 (SEQ ID NO:56) and CJ1-32; (SEQ ID NO: 57) CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQ IDNO: 35) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-31 (SEQ IDNO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQID NO: 35) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-22 (SEQID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-31 (SEQ ID NO: 56 and CJ1-32;(SEQ ID NO: 57) CJ1-1, (SEQ ID NO: 26) CJ1-2, (SEQ ID NO: 27) CJ1-16,(SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-22 (SEQ ID NO: 47) andCJ1-23; (SEQ ID NO: 48) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48)CJ1-24, (SEQ ID NO: 49) CJ1-9, (SEQ ID NO: 34) and CJ1-10; (SEQ ID NO:35) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQ ID NO:49) CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQ ID NO: 35) CJ1-16, (SEQ ID NO:41) and CJ1-17; (SEQ ID NO: 42) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ IDNO: 48) CJ1-24, (SEQ ID NO: 49) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ IDNO: 42) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-22, (SEQID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQ ID NO: 49) CJ1-16, (SEQID NO: 41) and CJ1-17; (SEQ ID NO: 42) CJ1-22, (SEQ ID NO: 47) CJ1-23,(SEQ ID NO: 48) CJ1-24, (SEQ ID NO: 49) CJ1-9, (SEQ ID NO: 34) CJ1-10,(SEQ ID NO: 35) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57)CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQ ID NO: 49)CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQ ID NO: 35) CJ1-16, (SEQ ID NO: 41)CJ1-17, (SEQ ID NO: 42) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO:57) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQ ID NO:49) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJI-42.5, (SEQ IDNO: 119) CJI-43.32, (SEQ ID NO: 125) CJI-43.39, (SEQ ID NO: 128)CJI-24.5 (SEQ ID NO: 129) and CJI-44.8; (SEQ ID NO: 132) CJI-42.5, (SEQID NO: 119) CJI-43.39, (SEQ ID NO: 128) CJI-24.5 (SEQ ID NO: 129) andCJI-44.8; (SEQ ID NO: 132) CJI-42.5, (SEQ ID NO: 119) CJI-43.39, (SEQ IDNO: 128) CJI-24.5 (SEQ ID NO: 129) and CJI-44.8; (SEQ ID NO: 132)CJI-42.5, (SEQ ID NO: 119) CJI-43.39 (SEQ ID NO: 128) and CJI-24.5; (SEQID NO: 129) CJI-42.5, (SEQ ID NO: 119) and CJI-43.39; (SEQ ID NO: 128)CJI-43.39, (SEQ ID NO: 128) CJI-24.5 (SEQ ID NO: 129) and CJI-44.8; (SEQID NO: 132) CJI-43.39 (SEQ ID NO: 128) and CJI-24.5; (SEQ ID NO: 129)CJI-43.39 (SEQ ID NO: 128) and CJI-44.8; (SEQ ID NO: 132) CJI-24.5, (SEQID NO: 129) CJI-44.8 (SEQ ID NO: 132) and CJI-42.5; (SEQ ID NO: 119)CJI-24.5 (SEQ ID NO: 129) and CJI-44.8; (SEQ ID NO: 132) CJI-44.8, (SEQID NO: 132) CJI-42.5 (SEQ ID NO: 119) and CJI-43.32; 125) CJI-44.8 (SEQID NO: 132) and CJI-42.5; (SEQ ID NO: 119) and CJI-44.8 (SEQ ID NO: 132)and CJI-43.32. (SEQ ID NO: 125)

Isolated Cry j I or Cry j II peptides within the scope of the inventioncan be used in methods of treating and preventing allergic reactions toJapanese cedar pollen. Thus, one aspect of the present inventionprovides therapeutic compositions comprising a peptide of Cry j I or Cryj II or a combination of peptides of both Cry j I or Cry j II, eachpeptide including at least one T cell epitope, and a pharmaceuticallyacceptable carrier or diluent. In another aspect, the therapeuticcomposition comprises a pharmaceutically acceptable carrier or diluentand a peptide comprising at least two regions, each region comprising atleast one T cell epitope of Cry j I or Cry j II.

Preferred therapeutic compositions comprise a sufficient percentage ofthe T cell epitopes of Cry j I or Cry j II or T cell epitopes of bothCry j I and Cry j II such that a therapeutic regimen of administrationof the composition to an individual sensitive to Japanese cedar pollenallergen, results in reduced T cell responsiveness. More preferably, thecomposition comprises a sufficient percentage of the T cell epitopessuch that at least about 40%, and more preferably at least about 60% ofthe T cell reactivity of Cry j I or Cry j II or both Cry j I or Cry j IIare included in the composition. Such compositions can be administeredto an individual to treat or prevent sensitivity to Japanese cedarpollen or to an allergen which is immunologically cross-reactive withJapanese cedar pollen allergen such as pollen from Jun s or Jun v.

In yet another aspect of the present invention, a composition isprovided comprising at least two peptides (e.g., a physical mixture ofat least two peptides), each comprising at least one T cell epitope ofCry j I or Cry j II. Such compositions can be administered in the formof a therapeutic composition with a pharmaceutically acceptable carrieror diluent. A therapeutically effective amount of one or more of suchcompositions can be administered simultaneously or sequentially to anindividual sensitive to Japanese cedar pollen. In another aspect of theinvention, Cry j I or Cry j II peptides are provided which can beadministered simultaneously or sequentially. Such combinations maycomprise therapeutic compositions composing only one peptide, or morepeptides if desired. Such compositions may be administeredsimultaneously or sequentially in preferred combinations.

Preferred compositions and preferred combinations of Cry j I peptideswhich can be administered simultaneously or sequentially (comprisingpeptides having amino acid sequences shown in FIG. 13 and FIG. 20)include the following combinations:

CJ1-1, (SEQ ID NO: 26) CJ1-2 (SEQ ID NO: 27) and CJ1-3; (SEQ ID NO: 28)CJ1-1 (SEQ ID NO: 26) and CJ1-2; (SEQ ID NO: 27) CJ1-9 (SEQ ID NO: 34)and CJ1-10; (SEQ ID NO: 35) CJ1-14, (SEQ ID NO: 39) CJ1-15, (SEQ ID NO:40) CJ1-16 (SEQ ID NO: 41) and CJ1-17; (SEQ ID NO: 42) CJ1-20, (SEQ IDNO: 45) CJ1-21, (SEQ ID NO: 46) CJ1-22 (SEQ ID NO: 47) and CJ1-23; (SEQID NO: 48 CJ1-20, (SEQ ID NO: 45) CJ1-22 (SEQ ID NO: 47) and CJ1-23;(SEQ ID NO: 48) CJ1-22 (SEQ ID NO: 47) and CJ1-23; (SEQ ID NO: 48)CJ1-22, (SEQ ID NO: 47) CJ1-23 (SEQ ID NO: 48) and CJ1-24; (SEQ ID NO:49) CJ1-24 (SEQ ID NO: 49) and CJ1-25; (SEQ ID NO: 50) CJ1-30, (SEQ IDNO: 55) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-31 (SEQID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-22, (SEQ ID NO: 47) CJ1-23,(SEQ ID NO: 48) CJ1-16 (SEQ ID NO: 41) and CJ1-17; (SEQ ID NO: 42)CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48 CJ1-31 (SEQ ID NO: 56)and CJ1-32; (SEQ ID NO: 57) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO:42) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-9, (SEQ IDNO: 34) CJ1-10 (SEQ ID NO: 35) and CJ1-16; (SEQ ID NO: 41) CJ1-16 (SEQID NO: 41) and CJ1-17; (SEQ ID NO: 42) CJ1-17, (SEQ ID NO: 42) CJ1-22(SEQ ID NO: 47) and CJ1-23; (SEQ ID NO: 48) CJ1-16, (SEQ ID NO: 41)CJ1-17 (SEQ ID NO: 42) and CJ1-20; (SEQ ID NO: 45) CJ1-31, (SEQ ID NO:56) CJ1-32 (SEQ ID NO: 57) and CJ1-20; (SEQ ID NO: 45) CJ1-22, (SEQ IDNO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-1, (SEQ ID NO: 26) CJ1-2 (SEQ ID NO:27) and CJ1-3; (SEQ ID NO: 28) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ IDNO: 42) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-31 (SEQ IDNO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQID NO: 35) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-22 (SEQID NO: 47) and CJ1-23; (SEQ ID NO: 48) CJ1-9, (SEQ ID NO: 34) CJ1-10,(SEQ ID NO: 35) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-31(SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-9, (SEQ ID NO: 34)CJ1-10, (SEQ ID NO: 35) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48)CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJ1-9, (SEQ ID NO:34) CJ1-10, (SEQ ID NO: 35) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO:42) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-31 (SEQ ID NO:56 and CJ1-32; (SEQ ID NO: 57) CJ1-1, (SEQ ID NO: 26) CJ1-2, (SEQ ID NO:27) CJ1-16, (SEQ ID NO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-22 (SEQ ID NO:47) and CJ1-23; (SEQ ID NO: 48) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ IDNO: 48) CJ1-24, (SEQ ID NO: 49) CJ1-9, (SEQ ID NO: 34) and CJ1-10; (SEQID NO: 35) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQID NO: 49) CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQ ID NO: 35) CJ1-16, (SEQID NO: 41) and CJ1-17; (SEQ ID NO: 42) CJ1-22, (SEQ ID NO: 47) CJ1-23,(SEQ ID NO: 48) CJ1-24, (SEQ ID NO: 49) CJ1-16, (SEQ ID NO: 41) CJ1-17,(SEQ ID NO: 42) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57)CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQ ID NO: 49)CJ1-16, (SEQ ID NO: 41) and CJ1-17; (SEQ ID NO: 42) CJ1-22, (SEQ ID NO:47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQ ID NO: 49) CJ1-9, (SEQ ID NO:34) CJ1-10, (SEQ ID NO: 35) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQ IDNO: 57) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQ IDNO: 49) CJ1-9, (SEQ ID NO: 34) CJ1-10, (SEQ ID NO: 35) CJ1-16, (SEQ IDNO: 41) CJ1-17, (SEQ ID NO: 42) CJ1-31 (SEQ ID NO: 56) and CJ1-32; (SEQID NO: 57) CJ1-22, (SEQ ID NO: 47) CJ1-23, (SEQ ID NO: 48) CJ1-24, (SEQID NO: 49) CJ1-31, (SEQ ID NO: 56) and CJ1-32; (SEQ ID NO: 57) CJI-42.5,(SEQ ID NO: 119) CJI-43.32, (SEQ ID NO: 125) CJI-43.39, (SEQ ID NO: 128)CJI-24.5 (SEQ ID NO: 129) and CJI-44.8; (SEQ ID NO: 132) CJI-42.5, (SEQID NO: 119) CJI-43.39, (SEQ ID NO: 128) CJI-24.5 (SEQ ID NO: 129) andCJI-44.8; (SEQ ID NO: 132) CJI-42.5, (SEQ ID NO: 119) CJI-43.39 (SEQ IDNO: 128) and CJI-24.5; (SEQ ID NO: 129) CJI-42.5, (SEQ ID NO: 119) andCJI-43.39; (SEQ ID NO: 128) CJI-43.39, (SEQ ID NO: 128) CJI-24.5 (SEQ IDNO: 129) and CJI-44.8; (SEQ ID NO: 132) CJI-43.39 (SEQ ID NO: 128) andCJI-24.5; (SEQ ID NO: 129) CJI-43.39 (SEQ ID NO: 128) and CJI-44.8; (SEQID NO: 132) CJI-24.5, (SEQ ID NO: 129) CJI-44.8 (SEQ ID NO: 132) andCJI-42.5; (SEQ ID NO: 119) CJI-24.5 (SEQ ID NO: 129) and CJI-44.8; (SEQID NO: 132) CJI-44.8, (SEQ ID NO: 132) CJI-42.5 (SEQ ID NO: 119) andCJI-43.32; (SEQ ID NO: 125) CJI-44.8 (SEQ ID NO: 132) and CJI-42.5; (SEQID NO: 119) and CJI-44.8 (SEQ ID NO: 132) and CJI-43.32. (SEQ ID NO:125)

Preferred compositions and preferred combinations of Cry j I peptideswhich can be administered simultaneously and/or sequentially may includeany of the above preferred Cry j I combinations and in addition, mayalso include compositions comprising at least one peptide, or acombination of peptides derived from Cry j II such as Cry j IIA (SEQ IDNO: 185), Cry j IIB (SEQ ID NO: 186), Cry j IIC (SEQ ID NO: 187), Cry jIID (SEQ ID NO: 188), Cry j IIE, (SEQ ID NO: 189) and Cry j IIF (SEQ IDNO: 190), Cry j IIG (SEQ ID NO: 191), Cry j IIH (SEQ ID NO: 192), andCry j IIQ (SEQ ID NO: 193) all as shown in FIG. 41.

Another aspect of this invention pertains to a multipeptide formulationsuitable for pharmaceutical administration to ragweed sensitiveindividuals. The multipeptide formulation includes at least two or morepeptides of Japanese cedar pollen protein allergen having human T cellstimulating activity in an in vitro T cell proliferation assay (i.e.,comprising at least one T cell epitope). Special considerations whenpreparing a multipeptide formulation include maintaining the solubilityand stability of all peptides in the formulation at a physiologicallyacceptable pH (e.g. . pH4-pH9 and even more preferably pH5.5-pH8.5).This requires choosing one or more pharmaceutically acceptable carrierssuch as excipients which are compatible with all the peptides in themultipeptide formulation. For example, suitable excipients includesterile water, sodium phosphate, mannitol or both sodium phosphate andmannitol or any combination thereof. Other suitable excipients includebut are not limited to sorbitol, sucrose, dextrose, lactose dextran andPVP. Additionally due to the potential for dimerization of the peptidesin a multipeptide formulation, there may also be included an agent suchas EDTA to prevent dimerization. Alternatively, any material orprocedures known in the art to prevent dimerization may be used. Inaddition, pharmaceutically acceptable counter ions may be added duringthe preparation of the multipeptide formulation. Examples ofpharmaceutically acceptable counter ions include acetate, HCl, andcitrate. A preferred multipeptide formulation includes at least twopeptides derived from Japanese cedar pollen protein allergen each havinghuman T cell stimulating activity and each soluble and stable at aphysiologically acceptable pH. In a preferred embodiment, themultipeptide formulation includes Cry j I peptides CJ1-24.5, CJ1-43.39and CJ1-44.8 and sodium phosphate and mannitol. For this embodiment, asuitable counter ion such as acetate may be added during the preparationof the formulation, and the formulation is preferably prepared in theform of a lyophilized powder which is reconstituted in a physiologicallyacceptable carrier, such as sterile water, prior to use. One,non-limiting example of a preferred multipeptide formulation of theinvention is described below. The Cry j I peptides CJ1-24.5, CJ1-43.39and CJ1-44.8 will preferably be combined during manufacturing with theappropriate counter ion to produce a vial containing a sterile, pyrogenfree, lyophilized powder having the following composition:

-   -   Active: Cry j I peptides CJ1-24.5, CJ1-43.39 and CJ1-44.8        -   In concentration of 7.5-1500 μg per peptide    -   Inactives: 0.05 M Sodium Phosphate pH 6.0-8.0        -   5% w/v Mannitol, U.S.P.    -   Diluent: Sterile Water for Injection, U.S.P. (initial        reconstitution)        -   0.9% Sodium Chloride for Injection (dilution beyond initial            reconstitution)            The multipeptide formulation of the invention can be            provided in the form of a kit, including instructions for            use.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1

Purification of Native Japanese Cedar Pollen Allergen (Cry j I)

The following is a description of the work done to biochemically purifythe major allergen, Cry j I in the native form. The purification wasmodified from published procedures (Yasueda et al., J. Allergy Clin.Immunol. 71:77, 1983).

100 g of Japanese cedar pollen obtained from Japan (Hollister-Stier,Spokane, Wash.) was defatted in 1 L diethyl ether three times, thepollen was collected after filtration and the ether was dried off in avacuum.

The defatted pollen was extracted at 4° C. overnight in 2 L extractionbuffer containing 50 mM tris-HCL, pH 7.8, 0.2 M NaCl and proteaseinhibitors in final concentrations: soybean trypsin inhibitor (2 μg/ml),leupeptin (1 μg/ml), pepstatin A (1 μg/ml) and phenyl methyl sulfonylfluoride (0.17 mg/ml). The insoluble material was reextracted with 1.2 Lextraction buffer at 4° C. overnight and both extracts were combinedtogether and depigmented by batch absorption with Whatman DE-52 DEAEcellulose (200 g dry weight) equilibrated with the extraction buffer.

The depigmented material was then fractionated by ammonium sulfateprecipitation at 80% saturation (4° C.), which removed much of the lowermolecular weight material. The resultant partially purified Cry j I waseither dialyzed in PBS buffer and used in T cell studies (see Example 6)or subjected to further purification (biochemically or by monoclonalaffinity chromatography) as described below.

The enriched Cry j I material was then dialyzed against 50 mMNa-acetate, pH 5.0 at 4° C. with 50 mM Na-acetate, pH 5.0 with proteaseinhibitors. The sample was next applied to a 100 ml DEAE cellulosecolumn (Whatman DE-52) equilibrated at 4° C. with 50 mM Na-acetate pH5.0 with protease inhibitors. The unbound material (basic proteins) wasthen applied to a 50 ml cation exchange column (Whatman CM-52) which wasequilibrated at 4° C. with 10 mM Na-acetate, pH 5.0 with proteaseinhibitors. Cry j I was eluted in the early fractions of a lineargradient 0.3 M NaCl. The enriched Cry j I material was lyophilized andwas then purified by FPLC over a 300 ml Superdex 75 column (Pharmacia)at a flow rate of 30 ml/h in 10 mM Na-acetate, pH 5.0 at 25° C.

The purified Cry j I was further applied to FPLC S-Sepharose 16/10column chromatography (Pharmacia) with a linear gradient of 0-1 M NaClat 25° C. Cry j I, eluted as the major peak, was subjected to a secondgel filtration chromatography. FPLC Superdex 75 column (2.6 by 60cm)(Pharmacia, Piscataway, N.J.) was eluted with a downward flow of 10mM Na-acetate, pH 5.0 with 0.15 M NaCl at a flow rate of 30 ml/h at 25°C. FIG. 1 a shows the chromatography on gel filtration. Only Cry j I wasdetected (FIG. 1 b, lane 2 to lane 8). Cry j I was fractionated into 3bands as analyzed by SDS-PAGE using silver staining (FIG. 1 b) As shownin FIG. 1 b, SDS PAGE (12.5%) analysis of the fractions from the majorpeak shown in FIG. 1 a was performed under reducing conditions. The gelwas silver stained using the silver staining kit from Bio-Rad. Thesamples in each lane were as follows: Lane 1, prestained standardproteins (Gibco BRL) including ovalbumin (43,000 kD), carbonic anhydrase(29,000 kD), and a-lactoglobulin (18,400 kD); lane 2, fraction 36; lane3 fraction 37; lane 4 fraction 38; lane 5 fraction 39 ; lane 6 fraction41, lane 7 fraction 43; and lane 8 fraction 44. All fractions are shownin FIG. 1 a.

These proteins were also analyzed by Western blotting using mousemonoclonal antibody CBF2 (FIG. 2). As shown in FIG. 2, an aliquot offraction 36 (lane 1), fraction 39, (lane 2) and fraction 43 (lane 3)purified from the Superdex 75 as shown in FIG. 1 was separated bySDS-PAGE, electroblotted onto nitrocelluslose and probed with mAB CBF2.Biotinlylated goat anti-mouse Ig was used for the second antibody andbound antibody was revealed by ¹²⁵I-streptavidin. The monoclonal CBF2was raised against ragweed allergen Amb a I by Dr. D. Klapper (ChapelHill, N.C.). Because of the homology between the Amb a I and Cry j Isequences, a number of antibodies raised against Amb a I were tested forreactivity with Cry j I. The results showed that CBF2 recognizeddenatured Cry j I as detected by ELISA and Western blotting. Inaddition, Western blotting also demonstrated that no other bands weredetected by CBF2, other than Cry j I in the expected molecular weightrange (FIG. 2). These results were consistent with the findings fromprotein sequencing. When fraction 44 and fraction 39 (FIG. 1 b) weresubjected to N-terminal sequencing, only Cry j I sequence was detected.

In summary, three Cry j I isoforms of different molecular weight werepurified from pollen extract. The molecular weights estimated bySDS-PAGE ranged from 40-35 kD under both reducing and non-reducingconditions. The isoelectric point of these isoforms is approximately9.5-8.6, with an average pI of 9.0. The N-terminal 20 amino acidsequence was the same in these 3 bands and was identical to previouslypublished Cry j I sequence (Taniai et al, supra). The 3 isoforms are allrecognized by monoclonal antibody CBF2 as shown in the allergic seratitration of different purified subfractions of Cry j I using a pool offifteen allergic patient plasma. They all bind allergic patient IgE(FIG. 3). The difference in molecular weight and isoelectric point inthese isoforms might in part be due to post-translational modification,e.g. glycosylation, phosphorylation or lipid content. The possibilitythat these different isoforms might be due to protease degradationcannot be ruled out at present even though it is unlikely due to thefact that four different protease inhibitors were used during extractionand purification. The other possibility could be due to polymorphism inthe gene or alternate splicing in the mRNA though only one major form ofCry j I protein has been detected in cDNA cloning studies (see Example4).

Another approach which may be used to purify native Cry j I orrecombinant Cry j I is immunoaffinity chromatography. This techniqueprovides a very selective protein purification due to the specificity ofthe interaction between monoclonal antibodies and antigen. For thepurpose of producing Cry j I-reactive monoclonal antibodies, femaleBalbl/c mice were obtained from Jackson Labs. Each mouse was initiallyimmunized intraperitoneally with 70-100 μg purified native Cry j I,(>99% purity lower band, as shown in FIG. 1 b), emulsified in Freund'scomplete adjuvant. One further intravenous injection of 10 μg purifiednative Cry j I in PBS was given 54 days after the initial injection. Thespleen was removed 3 days later and myeloma fusion was conducted asdescribed (Current Protocols in Immunology, 1991, Coligan et al, eds.)using the myeloma line SP2.0. The cells were cultured in 10% fetal calfserum (Hybrimax), hypoxanthine and azaserine and wells containingcolonies of hybridoma cells were screened for antibody production usingantigen-binding ELISA.

Cells from positive wells were cloned at three-tenths cell/well in 10%fetal calf serum (Hybrimax), hypoxanthine and positive clones weresubcloned one more time in hypoxanthine medium. Capture ELISA (seeExample 7) was used for secondary and tertiary screening. This assayoffers the advantage that a clone that recognizes the native protein maybe selected and thus may be useful for immunoaffinity purification. Forexample, two monoclonal antibodies (4B11. 8B11) were generated. Theseantibodies were purified by Gammabind G. Sepharose (Pharmacia,Piscataway, N.J.) according to manufacturer's procedures and wereimmobilized to cyanogen bromide—activated Sepharose 4B (Pharmacia,Piscataway, N.J.) according to the procedures described by Pharmacia.The ammonium sulphate preparation containing Cry j I was applied to theresin and unbound material was washed extensively with PBS. Cry j I waseluted with 2 column volumes of 0.1 M glycine, pH 2.7. Silver stainingof the eluate fractions run on SDS PAGE showed that Cry j I was purifiedalmost to homogeneity. These fractions did not contain detectable levelsof Cry j II. Other methods to immobilize MAb 8B11 were also tested.Similar results were obtained using purified MAb 8B11 covalentlycross-linked to Gammabind G Sepharose by dimethylpimelimidate (SchneiderC., et al, J. Biol. Chem. (1982) volume 257:10766-10769). However,experiments using purified MAb 8B11 covalently cross-linked to Affi-gel10 (Biorad, Richmond, Calif.) showed that although greater than 90% ofthe monoclonal antibody was covalently coupled to Affi-gel 10, the yieldof Cry j I purified over this resin was significantly less than thatpurified from MAb 8B11 cross-linked to cyanogen bromide-activatedSepharose 4B (data not shown). Nevertheless, the purified Cry j I fromthese monoclonal antibodies immobilized on different resins is stillintact and can be recognized by MAb 8B11 and 4B11 by capture ELISA.Thus, these MAbs will provide a useful tool in purification of Cry j Ifrom pollen extracts. Similarly, monoclonal antibodies that bind torecombinant Cry j I can also be used for immunoaffinity chromatography.In addition, the monoclonal antibodies generated may be useful fordiagnostic purposes. It may also be possible to raise different MAbsthat show some specificity towards these different isoforms of Cry j Iand thus would provide a useful tool to characterize these isoforms.

EXAMPLE 2

Attempted Extraction of RNA From Japanese Cedar Pollen

Multiple attempts were made to obtain RNA from commercially-available,non-defatted, Cryptomeria japonica (Japanese cedar) pollen (HollisterStier, Seattle, Wash.). Initially, the method of Sambrook et al.,Molecular Cloning. A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989) was used in which the sample wassuspended and lysed in 4 M guanidine buffer, ground under liquidnitrogen, and pelleted through 5.7 M cesium chloride byultracentrifugation. Various amounts (3, 5 and 10 g) of pollen invarying amounts of guanidine lysis buffer (10 and 25 ml) were tried.Centrifugation through cesium resulted in viscous material in the bottomof the tube, from which it was not possible to recover an RNA pellet.Although it was possible to obtain RNA from defatted Ambrosiaartemisiifolia (ragweed) pollen (Greer Laboratories, Lenior, N.C.) usingthis protocol, defatting the Cryptomeria japonica pollen with acetonebefore guanidine extraction also did not yield any RNA, as determined byabsorbance at A₂₆₀.

An acid phenol extraction of RNA according to the method in Sambrook etal., supra was attempted from Cryptomeria japonica pollen. The pollenwas ground and sheared in 4.5 M guanidine solution, acidified byaddition of 2 M sodium acetate, and extracted with water-saturatedphenol plus chloroform. After precipitation, the pellet was washed with4 M lithium chloride, redissolved in 10 mM Tris/5 mM EDTA/1% SDS,chloroform extracted, and re-precipitated with NaCl and absoluteethanol. It was possible to extract Ambrosia artemisiifolia but notCryptomeria japonica RNA with this procedure.

Next, 4 g of Cryptomeria japonica pollen was suspended in 10 mlextraction buffer (50 mM Tris, pH 9.0, 0.2 M NaCl, 10 mM Mg acetate anddiethylpyrocarbonate (DEPC) to 0.1%), ground in a mortar and pestle ondry ice, transferred to a centrifuge tube with 1% SDS, 10 mM EDTA and0.5% N-lauroyl sarcosine, and the mixture was extracted five times withwarm phenol. The aqueous phase was recovered after the finalcentrifugation, 2.5 vol. absolute ethanol was added, and the mixture wasincubated overnight at 4° C. The pellet was recovered by centrifugation,resuspended in 1 ml dH₂0 by heating to 65° C., and reprecipitated by theaddition of 0.1 vol. 3 M Na acetate and 2.0 vol. of ethanol. Nodetectable RNA was recovered in the pellet as judged by absorbance atA₂₆₀ and gel electrophoresis.

Finally, 500 mg of Cryptomeria japonica pollen was ground by mortar andpestle on dry ice and suspended in 5 ml of 50 mM Tris pH 9.0 with 0.2 MNaCl, 1 mM EDTA, 1% SDS that had been treated overnight with 0.1% DEPC,as previously described in Frankis and Mascarhenas (1980) Ann. Bot. 45:595-599. After five extractions with phenol/chloroform/isoamyl alcohol(mixed at 25:24:1), material was precipitated from the aqueous phasewith 0.1 volume 3 M sodium acetate and 2 volumes ethanol. The pellet wasrecovered by centrifugation, resuspended in dH₂0 and heated to 65° C. tosolubilize the precipitated material. Further precipitations withlithium chloride were not done. There was no detectable RNA recovered,as determined by absorbance at A₂₆₀ and gel electrophoresis.

In summary, it has not been possible to recover RNA from the commercialpollen. It is not known whether the RNA has been degraded during storageor shipment, or whether the protocols used in this example did not allowrecovery of extant RNA. However, RNA was recovered from freshCryptomeria japonica pollen and staminate cone samples. (See Example 3)

EXAMPLE 3

Extraction of RNA From Japanese Cedar Pollen and Staminate Cones andCloning of Cry j I

Fresh pollen and staminate cone samples, collected from a singleCryptomeria japonica (Japanese cedar) tree at the Arnold Arboretum(Boston, Mass.), were frozen immediately on dry ice. RNA was preparedfrom 500 mg of each sample, essentially as described by Frankis andMascarenhas, supra. The samples were ground by mortar and pestle on dryice and suspended in 5 ml of 50 mM Tris pH 9.0 with 0.2 M NaCl, 1 mMEDTA, 1% SDS that had been treated overnight with 0.1% DEPC. After fiveextractions with phenol/chloroform/isoamyl alcohol (mixed at 25:24:1),the RNA was precipitated from the aqueous phase with 0.1 volume 2 Msodium acetate and 2 volumes ethanol. The pellets were recovered bycentrifugation, resuspended in dH₂0 and heated to 65° C. for 5 min. Twoml of 4 M lithium chloride were added to the RNA preparations and theywere incubated overnight at 0° C. The RNA pellets were recovered bycentrifugation, resuspended in 1 ml dH₂0, and again precipitated with 3M sodium acetate and ethanol overnight. The final pellets wereresuspended in 100 μl dH₂0 and stored at −80° C.

First strand cDNA was synthesized from 8 μg flowerhead and 4 μg pollenRNA using a commercially available kit (cDNA synthesis systems kit, BRL,Gaithersburg, Md.) with oligo dT priming according to the method ofGubler and Hoffman (1983) Gene 25:263-269. An attempt was made toamplify cDNA encoding Cry j I using the degenerate oligonucleotide CP-1(which has the sequence 5′-GATAATCCGATAGATAG-3′(SEQ ID NO:3), wherein Tat position 3 can also be C; T at position 6 can also be C; G atposition 9 can also be A,T, or C; A at position 12 can also be T, or C;T at position 15 can also be C; A at position 16 can also be T; and G atposition 17 can also be C) and primers EDT and ED. Primer EDT has thesequence 5°-GGAATTCTCTAGACTGCAGGTTTTTTTTTTTTTTT-3′(SEQ ID NO: 24).Primer ED has the sequence 5′-GGAATTCTCTAGACTGCAGGT-3′ (SEQ ID NO: 23).CP-1 is the degenerate oligonucleotide sequence encoding the first sixamino acids of the amino terminus (AspAsnProlleAspSer (SEQ ID NO: 266),amino acids 1-6 of SEQ ID NO: 1) of Cry j I. EDT will hybridize with thepoly A tail of the gene. All oligonucleotides were synthesized byResearch Genetics, Inc. Huntsville, Ala. Polymerase chain reactions(FOR) were carried out using a commercially available kit (GeneAmp DNAAmplification kit, Perkin Elmer Cetus, Norwalk, Conn.) whereby 10 μl 10×buffer containing dNTPs was mixed with 1 μg of CP-1 and 1 μg of ED/EDTprimers (ED:EDT in a 3:1 M ratio), cDNA (3-5 μl of a 20 μl first strandcDNA reaction mix), 0.5 μl Amplitaq DNA polymerase, and distilled waterto 100 μl.

The samples were amplified with a programmable thermal controller (MJResearch, Inc., Cambridge, MA). The first 5 rounds of amplificationconsisted of denaturation at 94° C. for 1 minute, annealing of primersto the template at 45° C. for 1.5 minutes, and chain elongation at 70°C. for 2 minutes. The final 20 rounds of amplification consisted ofdenaturation as above, annealing at 55° C. for 1.5 minutes, andelongation as above. Five percent (5 μl) of this initial amplificationwas then used in a secondary amplification with 1 μg each of CP-2 (whichhas the sequence 5′-GGGAATTCAATTGGGCGCAGAATGG-3′ wherein T at position11 can also be C; G at position 17 can also be A, T, or C; G at position20 can also be A; T at position 23 can also be C; and G at position 24can also be C) (SEQ ID NO: 4), a nested primer, and ED, as above. Thesequence 5′-GGGAATTC-3′ (SEQ ID NO: 160) (bases 1 through 8 of SEQ IDNO: 4) in primer CP-2 represents an Eco RI site added for cloningpurposes; the remaining degenerate oligonucleotide sequence encodesamino acids 13-18 of Cry j I (AsnTrpAlaGlnAsnArg (SEQ ID NO: 267), aminoacids 13 through 18 of SEQ ID NO: 1). Multiple DNA bands were resolvedon a 1% GTG agarose gel (FMC, Rockport, ME), none of which hybridizedwith ³²P end- labeled probe CP-3 (SEQ ID NO: 5) in a Southern blotperformed according to the method in Sambrook et al. supra. Therefore,it was not possible to select a specific Cry j I DNA band and thisapproach was not pursued. CP-3 has the sequence5′-CTGCAGCCATTTTCIACATTAAA-3′ wherein A at position 9 can also be G; Tatposition 12 can also be C; A at position 18 can also be G; and A atposition 21 can also be G) (SEQ ID NO: 5). Inosine (I) is used atposition 15 in place of G or A or T or C to reduce degeneracy (Knoth etal. (1988) Nucleic Acids Res, 16: 10932). The sequence 5′-CTGCAG-3′(bases 1 through 6 of SEQ ID NO: 5) in primer CP-3 represent a Pst Isite added for cloning purposes; the remaining degenerateoligonucleotide sequence is the non-coding strand sequence correspondingto coding strand sequence encoding amino acids PheAsnValGluAsnGly (SEQID NO: 268) (amino acids 327 through 332 of SEQ ID NO: 1) from theinternal sequence of Cry jI.

A primary PCR was also performed on first-strand cDNA using CP-1 (SEQ IDNO: 3) and CP-3 (SEQ ID NO: 5), as above. A secondary PCR was performedusing 5% of the primary reaction using CP-2 (SEQ ID NO: 4) and CP-3 (SEQID NO: 5). Again, multiple bands were observed, none of which could bespecifically identified in a Southern blot as Cry j I, and this approachwas also not pursued.

Double-stranded cDNA was then synthesized from approximately 4 μg(pollen) or 8 μg (flowerhead) RNA using a commercially available kit(cDNA Synthesis System kit, BRL, Gaithersburg, MD). After a phenolextraction and ethanol precipitation, the cDNA was blunted with T4 DNApolymerase (Promega, Madison, WI), and ligated to ethanol precipitated,self-annealed, AT (SEQ ID NO: 20) and AL (SEQ ID NO: 22)oligonucleotides for use in a modified Anchored PCR reaction, accordingto the method in Rafnar et al. (1991) J. Biol. Chem. 266: 1229-1236;Frohman et al. (1990) Proc. Natl. Acad. Sci. USA 85: 8998-9002; and Rouxet al. (1990) BioTech. 8: 48-57. Oligonucleotide AT has the sequence5′-GGGTCTAGAGGTACCGTCCGATCGATCATT-3′ (SEQ ID NO: 20) (Rafnar et al.supra). Oligonucleotide AL has the sequence 5′-AATGATCGATGCT-3′ (SEQ IDNO: 22) (Rafnar et al. supra. The amino terminus of Cry j I wasamplified from the linkered cDNA (3 μl from a 20 μl reaction) with 1 μgeach of oligonucleotides AP (SEQ ID NO: 21) and degenerate Cry j Iprimer CP-7 (which has the sequence 5′-TTCATICGATTCTGGGCCCA-3′ wherein Gat position 8 can also be T; A at position 9 can also be G; C atposition 12 can also be T; and G at position 15 can also be A, T, orC)(SEQ ID NO: 6). Inosine (I) is used at position 6 in place of G or Aor T or C to reduce degeneracy (Knoth et al. supra). The degenerateoligonucleotide CP-7 (SEQ ID NO: 6) is the non-coding strand sequencecorresponding to coding strand sequence encoding amino acids 14-20(TrpAlaGlnAsnArgMetLys (SEQ ID NO: 269)) from the amino terminus of Cryj I (amino acids 14-20 of SEQ ID NO: 1). Oligonucleotide AP has thesequence 5′-GGGTCTAGAGGTACCGTCCG-3′ (SEQ ID NO: 21).

The primary PCR reaction was carried out as described herein. Fivepercent (5 μl) of this initial amplification was then used in asecondary amplification with 1 .μg each of AP (SEQ ID NO: 21) anddegenerate Cry j I primer CP-8 (SEQ ID NO: 7) an internally nested Cry jI oligonucleotide primer, as described herein. Primer CP-8 has thesequence 5′-CCTGCAGCGATTCTGGGCCCAAATT-3′ wherein G at position 9 canalso be T; A at position 10 can also be G; C at position 13 can also beT; G at position 16 can also be A, T, or C; and A at position 23 canalso be G)(SEQ ID NO: 7). The nucleotides 5′-CCTGCAG-3′ (bases 1 through7 of SEQ ID NO: 7) represent a Pst I restriction site added for cloningpurposes. The remaining degenerate oligonucleotide sequence is thenon-coding strand sequence corresponding to coding strand sequenceencoding amino acids 13-18 of Cry j I (AsnTrpAlaGlnAsnArg (SEQ ID NO:267), amino acids 13-18 of SEQ ID NO: 1) from the amino terminus of Cryj I. The dominant amplified product was a DNA band of approximately 193base pairs, as visualized on an ethidium bromide (EtBr)-stained 3% GTGagarose gel.

Amplified DNA was recovered by sequential chloroform, phenol, andchloroform extractions, followed by precipitation at −20° C. with 0.5volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol. Afterprecipitation and washing with 70% ethanol, the DNA was simultaneouslydigested with Xba I and Pst I in a 15 μl reaction and electrophoresedthrough a preparative 3% GTG NuSieve low melt gel (FMC, Rockport, Me.).The appropriate sized DNA band was visualized by EtBr staining, excised,and ligated into appropriately digested M13mp18 for sequencing by thedideoxy chain termination method (Sanger et al. (1977) Proc. Natl AcadSci. USA 74: 5463-5476) using a commercially available sequencing kit(Sequenase kit, U.S. Biochemicals, Cleveland, Ohio). It was initiallythought that ligatable material could only be derived from staminatecone-derived RNA. However, upon subsequent examination, it was shownthat ligatable material could be recovered from PCR product generatedfrom pollen-derived RNA, and from staminate cone-derived RNA.

The clone designated JC71.6 was found to contain a partial sequence ofCry j I. This was confirmed as an authentic clone of Cry j I by havingcomplete identity to the disclosed NH₂-terminal sequence of Cry j I(Taniai et al. supra). The amino acid at position 7 was determined to becysteine (Cys) in agreement with the sequence disclosed in U.S. Pat. No.4,939,239. Amino acid numbering is based on the sequence of the matureprotein; amino acid 1 corresponds to the aspartic acid (Asp) disclosedas the NH₂-terminus of Cry j I (Taniai et al. supra) The initiatingmethionine was found to be amino acid −21 relative to the first aminoacid of the mature protein. The position of the initiating methioninewas supported by the presence of upstream in-frame-stop codons and by78% homology of the surrounding nucleotide sequence with the plantconsensus sequence that encompasses the initiating methionine, asreported by Lutcke et al. (1987) EMBO J. 6:4348.

The cDNA encoding the remainder of Cry j I gene was cloned from thelinkered cDNA by using oligonucleotides CP-9 (which has the sequence5′ATGGATTCCCCTTGCTTA-3′)(SEQ ID NO: 8) and AP (SEQ ID NO: 21) in theprimary PCR reaction. Oligonucleotide CP-9 (SEQ ID NO: 8) encodes aminoacids MetAspSerProCysLeu (SEQ ID NO: 270) of Cry j I (amino acids −21through −16 of SEQ ID NO: 1) from the leader sequence of Cry j I, and isbased on the nucleotide sequence determined for the partial Cry j Iclone JC76.1.

A secondary PCR reaction was performed on 5% of the initialamplification mixture, with 1 μg each of AP (SEQ ID NO: 21) and CP-10(which has the sequence 5′-GGGAATTCGATAATCCCATAGACAGC-3′)(SEQ ID NO: 9),the nested primer. The nucleotide sequence 5′-GGGAATTC-3′ of primerCP-10 (bases 1 through 8 of SEQ ID NO: 9) represent an Eco RIrestriction site added for cloning purposes. The remainingoligonucleotide sequence encodes amino acids 1-6 of Cry j I(AspAsnProlleAspSer (SEQ ID NO: 266)) (amino acids 1 through 6 of SEQ IDNO: 1), and is based on the nucleotide sequence determined for thepartial Cry j I clone JC76.1. The amplified DNA product was purified andprecipitated as above, followed by digestion with Eco RI and Xba I andelectrophoresis through a preparative 1% low melt gel. The dominant DNAband was excised and ligated into M13 mp 19 and pUC19 for sequencing.Again, ligatable material was recovered from cDNA generated frompollen-derived RNA, and from staminate cone-derived RNA. Two clones,designated pUC19JC91a and pUC19JC91d, were selected for full-lengthsequencing. They were subsequently found to have identical sequences.

DNA was sequenced by the dideoxy chain termination method (Sanger et al.supra) using a commercially available kit (sequenase kit (U.S.Biochemicals, Cleveland, OH). Both strands were completely sequencedusing M13 forward and reverse primers (N.E. Biolabs, Beverly, MA) andinternal sequencing primers CP-13 (SEQ ID NO: 10), CP-14 (SEQ ID NO:11), CP-15 (SEQ ID NO: 12), CP-16 (SEQ ID NO: 13), CP-18 (SEQ ID NO:15), CP-19 (SEQ ID NO: 16), and CP-20 (SEQ ID NO: 17). CP-13 has thesequence 5′-ATGCCTATGTACATTGC-3′ (SEQ ID NO: 10). CP-13 (SEQ ID NO: 10)encodes amino acids 82-87 of Cry j I (MetProMetTyrlleAla (SEQ ID NO:271), amino acids 82 through 87 of SEQ ID NO: 1). CP-14 has the sequence5′-GCAATGTACATAGGCAT-3′ (SEQ ID NO: 11) and corresponds to thenon-coding strand sequence of CP-13 SEQ ID NO: 10). CP-15 has thesequence 5′-TCCAATTCTTCTGATGGT-3′ ((SEQ ID NO: 12) which encodes aminoacids 169-174 of Cry j I (SerAsnSerSerAspGly (SEQ ID NO: 272), aminoacids 169 through 174 of SEQ ID NO: 1). CP-16 has the sequence5′-TTTTGTCAATTGAGGAGT-3′ (SEQ ID NO: 13) which is the non-coding strandsequence which corresponds to coding strand sequence encoding aminoacids 335-340 of Cry j I (ThrProGlnLeuThrLys (SEQ ID. NO: 273), aminoacids 335 through 340 of SEQ ID NO: 1). CP-18 has the sequence5′-TAGCAACTCCAGTCGAAGT-3′ (SEQ ID NO: 15) which is the non-coding strandsequence which substantially corresponds to coding strand sequenceencoding amino acids 181 through 186 of Cry j I (ThrSerThrGlyValThr (SEQID NO: 274), amino acids 181 through 186 of SEQ ID NO: 1) except thatthe fourth nucleotide of CP-18 (SEQ ID NO: 15) was synthesized as a Crather than the correct nucleotide, T. CP-19 which has the sequence5′-TAGCTCTCATTTGGTGC-3′ (SEQ ID NO: 16) is the non-coding strandsequence which corresponds to coding strand sequence encoding aminoacids 270 through 275 of Cry j I (AlaProAsnGluSerTyr (SEQ ID NO: 275),amino acids 270 through 275 of SEQ ID NO: 1). CP-20 has the sequence5′-TATGCAATTGGTGGGAGT-3′ (SEQ ID NO: 17) which is the coding strandsequence for amino acids 251-256 of Cry j I (TyrAlalleGlyGlySer (SEQ IDNO: 276), amino acids 251 through 256 of SEQ ID NO: 1). The sequencedDNA was found to have the sequence shown in FIGS. 4 a and 4 b (SEQ IDNO: 1). This is a composite sequence from the two overlapping clones JC71.6 and pUC19J91a. The complete cDNA sequence for Cry j I is composedof 1312 nucleotides, including 66 nucleotides of 5′ untranslatedsequence, an open reading frame starting with the codon for aninitiating methionine, of 1122 nucleotides, and a 3′ untranslatedregion. There is a consensus polyadenylation signal sequence in the 3′untranslated region 25 nucleotides 5′ to the poly A tail (nucleotides1279-1283 of FIG. 4 and SEQ. ID NO: 1). Nucleotides 1313-1337 of FIG. 4and SEQ. ID NO: 1 represent vector sequences. The position of theinitiating methionine is confirmed by the presence of in-frame upstreamstop codons and by 78% homology with the plant consensus sequence thatencompasses the initiating methionine (AAAAAUGGA (bases 62 through 70 ofSEQ ID NO: 1)) found in Cry j I compared with the AACAAUGGC consensussequence for plants, Lutcke et al. (1987) EMBO J. 6:43-48). The openreading frame encodes a protein of 374 amino acids of which the first 21amino acids comprise a leader sequence that is cleaved from the matureprotein. The amino terminus of the mature protein was identified bycomparison with the published NH₂-terminal sequence (Taniai et al.(1988) supra) and with sequence determined by direct amino acid analysisof purified native Cry j I (Example 1). The deduced amino acid sequenceof the mature protein, comprised of 353 amino acids has completesequence identity with the published protein sequence for Cry j I(Taniai et al. supra), including the first twenty amino acids for theNH₂-terminal and sixteen contiguous internal amino acids. The matureprotein also contains five potential N-linked glycosylation sitescorresponding to the consensus sequence N-X-S/T.

EXAMPLE 4

Extraction of RNA from Japanese Cedar Pollen Collected in Japan

Fresh pollen collected from a pool of Cryptomeria japonica (Japanesecedar) trees in Japan was frozen immediately on dry ice. RNA wasprepared from 500 mg of the pollen, essentially as described by Frankisand Mascarenhas Ann. Bot. 45:595-599. The samples were ground by mortarand pestle on dry ice and suspended in 5 ml of 50 mM Tris pH 9.0 with0.2 M NaCl, 1 mM EDTA, 1% SDS that had been treated overnight with 0.1%DEPC. After five extractions with phenol/chloroform/isoamyl alcohol(mixed at 25:24:1), the RNA was precipitated from the aqueous phase with0.1 volume 3 M sodium acetate and 2 volumes ethanol. The pellets wererecovered by centrifugation, resuspended in dH₂0 and heated to 65° C.for 5 minutes. Two ml of 4 M lithium chloride were added to the RNApreparations and they were incubated overnight at 9° C. The RNA pelletswere recovered by centrifugation, resuspended in 1 ml dH₂0, and againprecipitated with 3 M sodium acetate and ethanol overnight. The finalpellets were resuspended in 100 μl dH₂0 and stored at −80° C.

Double stranded cDNA was synthesized from 8 μg pollen RNA using the cDNASynthesis Systems kit (BRL) with oligo dT priming according to themethod of Gubler and Hoffman (1983) Gene 25:263-269. Polymerase chainreactions (PCR) were carried out using the GeneAmp DNA Amplification kit(Perkin Elmer Cetus) whereby 10 μl 10× buffer containing dNTPs was mixedwith 100 pmol each of a sense oligonucleotide and an anti-senseoligonucleotide, (10 μl of a 400 μl double stranded cDNA reaction mix),0.5 μl. Amplitaq DNA polymerase, and distilled water to 100 μl.

The samples were amplified with a programmable thermal controller fromMJ Research, Inc. (Cambridge, Mass.). The first 5 rounds ofamplification consisted of denaturation at 94° C. for 1 minute,annealing of primers to the template at 45° C. for 1 minute, and chainelongation at 72° C. for 1 minute. The final 20 rounds of amplificationconsisted of denaturation as above, annealing at 55° C. for 1 minute,and elongation as above.

Seven different Cry j I primer pairs were used to amplify the doublestranded cDNA as follows: CP-9 (SEQ ID NO: 8) and CP-17 (SEQ ID NO: 14),CP-10 (SEQ ID NO: 9) and CP-17 (SEQ ID NO: 14), CP-10 (SEQ ID NO: 9) andCP-16 (SEQ ID NO: 13), CP-10 (SEQ ID NO: 9) and CP-19 (SEQ ID NO: 16),CP-10 (SEQ ID NO: 9) and CP-18 (SEQ ID NO: 15), CP-13 (SEQ ID NO: 10)and CP-17 (SEQ ID NO: 14), and CP-13 (SEQ ID NO: 10) and CP-19 (SEQ IDNO: 16). CP-17 has the sequence 5′-CCTGCAGAAGCTTCATCAACAACGTTTAGA-3′(SEQ ID NO: 14) and corresponds to non-coding strand sequence thatcorresponds to coding strand sequence encoding amino acids SKRC* (SEQ IDNO: 277) (amino acids 350-353 and the stop codon of SEQ ID NO: 1). Thenucleotide sequence 5′-CCTGCAGAAGCTT-3′ (SEQ ID NO: 278) (bases 1through 13 of SEQ ID NO: 14) represents Pst I and Hin dill restrictionsites added for cloning purposes. The nucleotide sequence 5′-TCA-3′(bases 13 through 15 of SEQ ID NO: 14) correspond to the non-codingstrand sequence of a stop codon. All of the amplifications yieldedproducts of the expected size when viewed on ethidium bromide(EtBr)-stained agarose gels. Two of these primer pairs were used inamplifications whose products were cloned into pUC19 for full-lengthsequencing. The PCR reaction with CP-10 (SEQ ID NO: 9) and CP-16 (SEQ IDNO: 13) on the double stranded cDNA yielded a band of approximately 1.1kb, and was called JC130. A separate first strand cDNA reaction was donewith 8 pg pollen RNA as described above and amplified witholigonucleotide primers CP-10 (SEQ ID NO: 9) and CP-17 (SEQ ID NO: 14).This amplification yielded a full-length cDNA, named JC135, from theamino terminus of the mature protein to the stop codon.

Amplified DNA was recovered by sequential chloroform, phenol, andchloroform extractions, followed by precipitation at −20° C. with 0.5volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol. Afterprecipitation and washing with 70% ethanol, the DNA was blunted with T4polymerase followed by digestion with Eco RI, in the case of JC130, orsimultaneously digested with Eco RI and Pst I, in the case of JC135, ina 15 μl reaction and electrophoresed through a preparative 1% SeaPlaquelow melt gel (FMC). Appropriate sized DNA bands were visualized by EtBrstaining, excised, and ligated into appropriately digested pUC19 fordideoxy DNA sequencing by the dideoxy chain termination method (Sangeret al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5476) using acommercially available sequencing kit (Sequenase kit, U.S. Biochemicals,Cleveland, Ohio).

Both strands were sequenced using M13 forward and reverse primers (N.E.Biolabs, Beverly, Mass.) and internal sequencing primers CP-13 (SEQ IDNO: 10), CP-15 (SEQ ID NO: 12), CP-16 (SEQ ID NO: 13), CP-18 (SEQ ID NO:15), CP-19 (SEQ ID NO: 16) and CP-20 (SEQ ID NO: 17). Two clones fromamplification JC130 (JC130a and JC130b) and one clone from amplificationJC135 (JC135g) were found to be Cry j I clones upon sequencing. Thenucleotide and deduced amino acid sequences of clones

JC130a and JC135g were identical to previously known Cry j I sequence(SEQ ID NO: 1). Clone JC130b was found to contain a single nucleotidedifference from the previously known Cry j I sequence (SEQ ID NO: 1).Clone JC130b had a C at nucleotide position 306 of SEQ ID NO: 1. Thisnucleotide change results in a predicted amino acid change from a Tyr toa His at amino acid 60 of the mature Cry j I protein. This polymorphismhas not yet been confirmed in an independently-derived PCR clone or bydirect amino acid sequencing. However, such polymorphisms in primarynucleotide and amino acid sequences are expected.

EXAMPLE 5

Expression of Cry j I

Expression of Cry j I was performed as follows. Ten μg of pUC19JC91a wasdigested with Xba I, precipitated, then blunted with T4 polymerase. BamHI linkers (N.E. Biolabs, Beverly, Mass.) were blunt-end ligated topUC19JC91a overnight and excess linkers were removed by filtrationthrough a NACS ion exchange minicolumn (BRL, Gaithersburg, Md.). TheTinkered cDNA was then digested simultaneously with EcoR I and BamH I.The Cry j I insert (extending from the nucleotides encoding the aminoterminus of the mature protein through the stop codon) was isolated byelectrophoresis of this digest through a 1% SeaPlaque low melt agarosegel. The insert was then ligated into the appropriately digestedexpression vector pET-11d (Novagen, Madison, Wis.; Jameel et al. (1990)J. Virol. 64:3963-3966) modified to contain a sequence encoding 6histidines (His 6) immediately 3′ of the ATG initiation codon followedby a unique EcoR I endonuclease restriction site. A second EcoR Iendonuclease restriction site in the vector, along with neighboring ClaI and Hind III endonuclease restriction sites, had previously beenremoved by digestion with EcoR I and Hind III, blunting and religation.

The histidine (His6) sequence was added for affinity purification of therecombinant protein (Cry j I) on a Ni²⁺ chelating column (Hochuli et al.(1987) J. Chromatog. 411:177-184; Hochuli et al. (1988) Bio/Tech.6:1321-1325.). A recombinant clone was used to transform Escherichiacoli strain BL21-DE3 which harbors a plasmid that has anisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter precedingthe gene encoding T7 polymerase. Induction with IPTG leads to highlevels of 17 polymerase expression, which is necessary for expression ofthe recombinant protein in pET-11d, which has a T7 promoter. ClonepET-11dΔHRhis₆JC91a.d was confirmed by dideoxy sequencing (Sanger et al.supra) with CP-14 (SEQ ID NO: 11) to be a Cry j I clone in the correctreading frame for expression.

Expression of the recombinant protein was confirmed in an initial smallculture (50 ml). An overnight culture of clone pET-11dΔHRhis₆JC91a.d wasused to inoculate 50 ml of media (Brain Heart Infusion Media, Difco)containing ampicillin (200 μg/ml), grown to an A₆₀₀=1.0 and then inducedwith IPTG (1 mM, final concentration) for 2 hrs. One ml aliquots of thebacteria were collected before and after induction, pelleted bycentrifugation, and crude cell lysates prepared by boiling the pelletsfor 5 minutes in 50 mM Tris HCl, pH 6.8, 2 mM EDTA, 1% SDS, 1%β-mercaptoethanol, 10% glycerol, 0.25% bromophenol blue (Studier et al.,(1990) Methods in Enzymology 185:60-89). Recombinant protein expressionwas visualized as a band with the predicted molecular weight ofapproximately 38 kDa on a Coomassie blue-stained SDS-PAGE gel, accordingto the method in Sambrook et al., supra, on which 40 μl of the crudelysate was loaded. A negative control consisted of crude lysates fromuninduced bacteria containing the plasmid with Cry j I and an inducedlysate from bacteria carrying no plasmid.

The pET-11dΔHRhis₆JC91a.d clone was then grown on a large scale forrecombinant protein expression and purification. A 2 ml culture bacteriacontaining the recombinant plasmid was grown for 8 hr, then streakedonto solid media (e.g. 6 petri plates (100×15 mm) with 1.5% agarose inLB medium (Gibco-BRL, Gaithersburg, Md.) containing 200 μg/mlampicillin), grown to confluence overnight, then scraped into 9 L ofliquid media (Brain Heart Infusion media, Difco) containing ampicillin(200 μg/ml). The culture was grown until the A₆₀₀ was 1.0, IPTG added (1mM final concentration), and the culture grown for an additional 2hours.

Bacteria were recovered by centrifugation (7,930×g, 10 min), and lysedin 90 ml of 6M Guanidine-HCl, 0.1M Na₂HPO₄, pH 8.0 for 1 hour withvigorous shaking. Insoluble material was removed by centrifugation(11,000×g, 10 min, 4° C.). The pH of the lysate was adjusted to pH 8.0,and the lysate applied to an 80 ml Nickel NTA agarose column (Qiagen)that had been equilibrated with 6 M Guanidine HCl, 100 mM Na₂HPO₄, pH8.0. The column was sequentially washed with 6 M Guanidine HCl, 100 mMNa₂HPO₄, 10 mM Tris-HCl, pH 8.0, then 8 M urea, 100 mM Na₂HPO₄, pH 8.0,and finally 8 M urea, 100 mM sodium acetate, 10 mM Tris-HCl, pH 6.3. Thecolumn was washed with each buffer until the flow through had anA₂₈₀≦0.05.

The recombinant protein, Cry j I, was eluted with 8 M u.ea, 100 mMsodium acetate, 10 mM Tris-HCl, pH 4.5, and collected in 10 ml aliquots.The protein concentration of each fraction was determined by absorbanceat A₂₈₀ and the peak fractions pooled. An aliquot of the collectedrecombinant protein was analyzed on SDS-PAGE according to the method inSambrook et al., supra.

The first 9 L prep, JCpET-1, yielded 30 mg of Cry j I with approximately78% purity, as determined by densitometry (Shimadzu Flying Spot Scanner,Shimadzu Scientific Instruments, Inc., Braintree, Mass.) of theCoomassie-blue stained SDS-PAGE gel. A second 9 L prep prepared the sameway, JCpET-2, yielded 41 mg of Cry j I with approximately 77% purity.

EXAMPLE 6

Japanese Cedar Pollen Allergic Patient T Cell Studies with Cry j I—thePrimary Cedar Pollen Antigen.

Synthesis of Overlapping Peptides

Japanese cedar pollen Cry j I overlapping peptides were synthesizedusing standard Fmoc/tBoc synthetic chemistry and purified by ReversePhase HPLC. FIG. 13 shows Cry j I peptides used in these studies. Thepeptide names are consistent throughout.

T Cell Responses to Cedar Pollen Antigenic Peptides

Peripheral blood mononuclear cells (PBMC) were purified by lymphocyteseparation medium (LSM) centrifugation of 60 ml of heparinized bloodfrom Japanese cedar pollen-allergic patients who exhibited clinicalsymptoms of seasonal rhinitis and were MAST and/or skin test positivefor Japanese cedar pollen. Long term T cell lines were established bystimulation of 2×10⁶ PBL/ml in bulk cultures of complete medium(RPMI-1640, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 5×10⁻⁵M2-mercaptoethanol, and 10 mM HEPES supplemented with 5% heat inactivatedhuman AB serum) with 20 μg/ml of partially purified native Cry j I (75%purity containing three bands similar to the three bands in FIG. 2) for7 days at 37° C. in a humidified 5% CO₂ incubator to select for Cry j Ireactive T cells. This amount of priming antigen was determined to beoptimal for the activation of T cells from most cedar pollen allergicpatients. Viable cells were purified by LSM centrifugation and culturedin complete medium supplemented with 5 units recombinant human IL-2/mland 5 units recombinant human IL-4/ml for up to three weeks until thecells no longer responded to lymphokines and were considered “rested”.The ability of the T cells to proliferate to selected peptides,recombinant Cry j I (rCry j I), purified native Cry j I, or recombinantAmb aI.1 (rAmb aI.1) or a positive control, phyto-hemaglutinin (PHA) wasthen assessed. For assay, 2×10⁴ rested cells were restimulated in thepresence of 2×10⁴ autologous Epstein-Barr virus (EBV)-transformed Bcells (prepared as described below) (gamma-irradiated with 25,000 RADS)with 2-50 μg/ml of selected peptides, Cry j I, purified native Cry j Ior rAmb a I.1 or PHA, in a volume of 200 μl complete medium in duplicateor triplicate wells in 96-well round bottom plates for 2-4 days. Theoptimal incubation was found to be 3 days. Each well then received 1 μCitritiated thymidine for 16-20 hours. The counts incorporated werecollected onto glass fiber filter mats and processed for liquidscintillation counting. FIG. 12 shows the effect of varying antigen dosein assays with recombinant Cry j I, purified native Cry j I, andrecombinant Amb a I.1 and several antigenic peptides synthesized asdescribed above. Some peptides were found to be inhibitory at highconcentrations in these assays. The titrations were used to optimize thedose of peptides in T cell assays. The maximum response in a titrationof each peptide is expressed as the stimulation index (S.I.). The S.I.is the counts per minute (CPM) incorporated by cells in response topeptide, divided by the CPM incorporated by cells in medium only. AnS.I. value equal to or greater than 2 times the background level isconsidered “positive” and indicates that the peptide contains a T cellepitope. The positive results were used in calculating mean stimulationindices for each peptide for the group of patients tested. The resultsshown in FIG. 12 demonstrate that patient #999 responds well torecombinant Cry j I (SEQ ID NO: 1), and purified native Cry j I, as wellas to peptides CJ1-2 (SEQ ID NO: 27), 3 (SEQ ID NO: 28), 20 (SEQ ID NO:45), and 22 (SEQ ID NO: 47) but not to recombinant Amb a I.1. Thisindicates that Cry j I T cell epitopes are recognized by T cells fromthis particular allergic patient and that rCry j I and peptides (SEQ IDNO: 27), 3 (SEQ ID NO: 28), 20 (SEQ ID NO: 45), and 22 (SEQ ID NO: 47)contain such T cell epitopes. Furthermore, the epitopes were often notdetected with the adjacent overlapping peptides, and therefore probablyspan the non-overlapping central residues of the reactive peptides. Nosignificant cross-reactivity was found in T cell assays using T cellsprimed with control antigens or with Cry j I primed T cells againstother antigens.

The above procedure was followed with a number of other patients.Individual patient results were used in calculating the mean S.I. foreach peptide if the patient responded to the Cry j I protein at an S.I.of 2.0 or greater and the patient responded to at least one peptidederived from Cry j I at an S.I. of 2.0 or greater. A summary of positiveexperiments from twenty-five patients is shown in FIG. 14. The barsrepresent the positivity index. Above each bar is the percent ofpositive responses with an S.I. of at least two to the peptide orprotein in the group of patients tested. In parenthesis above each barare the mean stimulation indices for each peptide or protein for thegroup of patients tested. All twenty-five T cell lines responded topurified native Cry j I and 68.0% of the T cell lines responded to rCryj I. These twenty-five T cell lines also responded at a significantlylower level to rAmb a I.1 indicating that the Amb a I allergens share adegree of homology with Cry j I and that “shared” T cell epitopes mightexist between Cry j I and Amb a I. This panel of Japanese cedar allergicpatients responded to peptides CJ1-1 (SEQ ID NO: 26), CJ1-2 (SEQ ID NO:27), CJ1-3 (SEQ ID NO: 28), CJ1-4 (SEQ ID NO: 29), CJ1-7 (SEQ ID NO:32), CJ1-8 (SEQ ID NO: 33), CJ1-9 (SEQ ID NO: 34), CJ1-10 (SEQ ID NO:35), CJ1-11 (SEQ ID NO: 36), CJ1-12 (SEQ ID NO: 37), CJ1-14 (SEQ ID NO:39), CJ1-15 (SEQ ID NO: 40), CJ1-16 (SEQ ID NO: 41), CJ1-17 (SEQ ID NO:42), CJ1-18 (SEQ ID NO: 43), CJ1-19 (SEQ ID NO: 44), CJ1-20 (SEQ ID NO:45), CJ1-21 (SEQ ID NO: 46), CJ1-22 (SEQ ID NO: 47), CJ1-23 (SEQ ID NO:48), CJ1-24 (SEQ ID NO: 49), CJ1-25 (SEQ ID NO: 50), CJ1-26 (SEQ ID NO:51), CJ1-27 (SEQ ID NO: 52), CJ1-28 (SEQ ID NO: 53), CJ1-30 (SEQ ID NO:55), CJ1-31 (SEQ ID NO: 56), CJ1-32 (SEQ ID NO: 57), CJ1-33 (SEQ ID NO:58), CJ1-34 (SEQ ID NO: 59) and CJ1-35 (SEQ ID NO: 60) indicating thatthese peptides contain T cell epitopes.

Preparation of (EBV)-Transformed B Cells for Use as Antigen PresentingCells

Autologous EBV-transformed cell lines were y-irradiated with 25,000 Radand used as antigen presenting cells in secondary proliferation assaysand secondary bulk stimulations. These EBV-transformed cell lines weremade by incubating 5×10⁶ PBL with 1 ml of B-59/8 Marmoset cell line(ATCC CRL1612, American Type Culture Collection, Rockville, Md.)conditioned medium in the presence of 1 μg/ml phorbol 12-myristate13-acetate (PMA) at 37° C. for 60 minutes in 12×75 mm polypropyleneround-bottom Falcon snap cap tubes (Becton Dickinson Labware, LincolnPark, N.J.). These cells were then diluted to 1.25×10⁶ cells/ml inRPMI-1640 as described above except supplemented with 10%heat-inactivated fetal bovine serum and cultured in 200 μl aliquots inflat bottom culture plates until visible colonies were detected. Theywere then transferred to larger wells until the cell lines wereestablished.

EXAMPLE 7

Cry j I as the Major Cedar Pollen Allergen

To examine the importance of Cry j I, reported as the major allergen ofJapanese cedar pollen, both direct and competition ELISA assays wereperformed. For the direct ELISA assays, wells were coated with eithersoluble pollen extract (SPE) of Japanese cedar pollen or purified nativeCry j I (assayed at 90% purity by protein sequencing) and human IgEantibody binding to these antigens was analyzed. Pooled human plasma,consisting of an equal volume of plasma from 15 patients with a Japanesecedar pollen MAST score of 2.5 or greater, and two individual patientplasma samples were compared in this assay. FIG. 5 shows the results ofthe binding reactivity with these two antigens. The overall pattern ofbinding is very similar whether the coating antigen is SPE (FIG. 5 a) orpurified native Cry j I (FIG. 5 b).

In the competition assay, ELISA wells were coated with Japanese cedarpollen SPE and then allergic patient IgE binding was measured in thepresence of competing purified native Cry j I in solution. The source ofallergic IgE in these assays was either the pool of plasma from 15patients (denoted PHP) or seven individual plasma samples from patientswith a Japanese cedar MAST score of 2.5 or greater. The competitionassay using the pooled human plasma samples compares the competitivebinding capacity of purified native Cry j I to Japanese cedar pollen SPEand an irrelevant allergen source, rye grass SPE. FIG. 6 shows thegraphed results of the competition ELISA with pooled human plasma. Theconcentration of protein present in the Japanese cedar pollen SPE isapproximately 170 times greater at each competing point than is thepurified native Cry j I. From this analysis it is clear that thepurified native Cry j I competes very well for IgE binding to the wholerange of proteins present in the Japanese cedar pollen soluble pollenextract. This implies that most of the anti-Cry j IgE reactivity isdirected against native Cry j I. The negative control shows no specificcompetitive activity and the competing SPE in solution can completelyremove binding to the coated wells. This assay was repeated withindividual patients as a measure of the range of the IgE response withinthe allergic population. FIG. 7 shows this result where the competitionof binding to SPE was performed with purified native Cry j I. Theresults demonstrate that although the patients show different doseresponse to Japanese cedar pollen SPE, each of the seven patients' IgEbinding to Japanese cedar pollen SPE could be competed with purifiednative Cry j I. The implications of these data are that for each patientthe IgE reactivity directed against Cry j I is predominant but thatthere is variation in this reactivity between patients. The overallconclusion is that these data support the previous findings (Yasueda etal., (1988) supra) that Cry j I is the major allergen of Japanese cedarpollen.

The reactivity of IgE from cedar pollen allergic patients to the pollenproteins is dramatically reduced when these proteins are denatured. Onemethod of analyzing this property is through direct binding ELISA wherethe coating antigen is the Japanese cedar pollen SPE or denaturedJapanese cedar pollen SPE which has been denatured by boiling in thepresence of a reducing agent DTT. This is then examined with allergicpatient plasma for IgE binding reactivity. FIG. 8 a, shows the directbinding assay to the SPE with seven individual plasma samples. In FIG. 8b, the binding results with the denatured SPE demonstrates the markeddecrease in reactivity following this treatment. To determine the extentof Cry j I binding to the ELISA wells, Cry j I was detected with arabbit polyclonal antisera against the Amb a I & II protein family.These ragweed proteins have high sequence identity (46%) with Cry j Iand this antisera can be used as a cross reactive antibody detectionsystem. In conclusion, these data demonstrate a marked loss in IgEreactivity following denaturation of the Japanese cedar pollen SPE.

EXAMPLE 8

IgE Reactivity and Histamine Release Analysis

The recombinant Cry j I protein (rCry j I), expressed in bacteria andthen purified (as described in Example 5), has been examined for IgEreactivity. The first method applied to this examination was directELISA where wells were coated with the recombinant Cry j I and IgEbinding was assayed on individual patients. FIG. 9 is the graphicrepresentation of this direct ELISA. The only positive signals on thisdata set are from the two control antisera rabbit polyclonal anti-Amb aI & II prepared by conventional means (Rabbit anti-Amb a I & II) andCBE2, a monoclonal antibody raised against Amb a I that cross reactswith Cry j I. By this method all patients tested showed no IgEreactivity with the recombinant Cry j I.

Another method of analysis that was applied to the examination of IgEreactivity to the recombinant Cry j I was a capture ELISA. This analysisrelies on the use of a defined antibody, in this case CBF2 to bind theantigen and allow for the binding of antibodies to other epitope sites.The format of this capture ELISA is 1) wells are coated with MAb CBF2,2) antigen or PBS (as one type of negative control) is added andcaptured by specific interaction with the coated MAb, 3) either thecontrol antibody anti-Amb a I & II (FIG. 10 b) or human allergic plasma(FIG. 10 a) is added as the detecting antibody, and 4) detection ofantibody binding is assayed. FIGS. 10 a and 10 b are the graphed resultsof these assays. For the IgE analysis, the pooled human plasma (PHP) (15patients) was used. The conclusion from these results is that there isno indication of any specific binding of human allergic IgE to rCry j Iby this method of analysis. However, the capture of rCry j I works asevidenced by the control antibody binding curve, shown in FIG. 10 b. Thelack of IgE binding to E. coli expressed rCry j I may be due to absenceof carbohydrate or any other post-translational modification and/or thatthe majority of IgE cannot react with denatured Cry j I. RAST,competition ELISA and Western blotting data also demonstrates nospecific IgE reactivity to the rCry j I (data not shown).

A histamine release assay was performed on one Japanese cedar pollenallergic patient using Japanese cedar pollen SPE, purified native Cry jI and rCry j I as the added antigens. This assay is a measure of IgEreactivity through human basophil mediator release. The results of thisassay, shown in FIG. 11, demonstrate strong histamine release with bothpurified native Cry j I and the Japanese cedar pollen SPE over a wideconcentration range. The only point where there is any measurablehistamine release with the Cry j I is at the highest concentration, 50μg/ml. Two possible explanations for this release by the rCry j Iare: 1) specific reactivity with a very low proportion of the anti-Cry jI IgE capable of recognizing the recombinant form of Cry j I, or 2)non-specific release caused by low abundance of bacterial contaminantsobserved only at the highest antigen concentration. Thus far, thisresult has only been shown in a single patient. In addition, the datashown are from single data points at each protein concentration.

It may be possible to use this recombinantly expressed Cry j I proteinfor immunotherapy as E. coli expressed material has T cell reactivity(Example 6), but does not appear to bind IgE from Crytpomeria japonicaatopes nor cause histamine release from the mast cells and basophils ofsuch atopes in vitro. Expression of rCry j I which is capable of bindingIgE could possibly be achieved in yeast, insect (baculovirus) ormammalian cells (e.g. CHO, human and mouse). A specific example ofmammalian cell expression could be the use of the pcDNA l/Amp mammalianexpression vector (Invitrogen, San Diego, Calif.) expressing recombinantCry j I in COS cells. A rCry j I capable of actively binding IgE may beimportant for the use of recombinant material for diagnostic purposes.

To analyze IgE reactivity to selected Cry j I peptides a direct ELISAformat was used. ELSIA wells were coated with 25 peptides derived fromCry j I and assayed for IgE binding. FIG. 15 a and 15 b are graphs ofthese binding results using PHP (15 patients) as the cedar pollenallergic IgE source. This pool of plasma was formulated for enrichmentof IgE that could bind to denatured SPE (as determined by direct ELISA)and therefore increase the chance of reactivity toward the peptides. Inthis assay, the peptide IgE binding capacity was compared to that ofpurified native Cry j I and to rCry j I. The only specific IgE detectedin this assay was to purified native Cry j I which supports the findingthat Japanese cedar allergic patient IgE does not bind to recombinantCry j I or the recombinant Cry j I peptides tested (FIG. 15).

EXAMPLE 9

Extraction of RNA from Juniperus sabinoides, Juniperus virginana andCupressus arizonica Pollens and the Cloning of Jun s I and Jun v I,Homologs of Cry j I.

Fresh pollen was collected from a single Juniperus virginiana tree atthe Arnold Arboretum (Boston, Mass.), and was frozen immediately on dryice; Juniperus sabinoides and Cupressus arizonica pollens were purchasedfrom Greer Laboratories, Inc. (Lenoir, N.C.). Total RNA was preparedfrom J. virginiana, J. sabinoides, and C. arizonica pollens as describedin Example 3. Single stranded cDNA was synthesized from 5 μg totalpollen RNA from J. virginiana and 5 μg total pollen RNA from J.sabinoides using the cDNA Synthesis System kit (BRL, Gaithersburg, Md.),as described in Example 3.

The initial attempt at cloning Cry j I homologue from the two juniperspecies was made using various pairs of Cry j I-specificoligonucleotides in PCR amplifications on both juniper cDNAs. PCRs werecarried out as described in Example 3. The oligonucleotide primer pairsused were: CP-9 (SEQ ID NO: 8)/CP-17 (SEQ ID NO: 14), CP10 (SEQ ID NO:9)/CP-17 (SEQ ID NO: 14), CP-10 (SEQ ID NO: 9)/CP-16 (SEQ ID NO: 13),CP-1O (SEQ ID NO: 9)/CP-19 (SEQ ID NO: 16), CP-10 (SEQ ID NO: 9)/CP-18(SEQ ID NO: 15), CP-13 (SEQ ID NO: 13)/CP-17 (SEQ ID NO: 14), and CP-13(SEQ ID NO: 10)/CP-19. CP-10 (SEQ ID NO: 9) was used in the majority ofthe reactions as the 5′ primer since it has been reported by Gross et.al. (1978) Scand. J. Immunol. 8: 437-441 that the first 5 amino-terminalamino acids of J. sabinoides are identical to those of Cry j I. Theseoligonucleotides and oligonucleotide primers pairs are described inExample 3. None of the primer pairs cited above resulted in a PCRproduct for either juniperus species when viewed on an EtBr-stained 1%agarose (FMC Bioproducts, Rockland, Me.) minigel.

The next series of PCR amplifications attempting to clone the Cry j Ihomologues from J. sabinoides and J. virginiana from were made on doublestranded linkered cDNA synthesized from RNA from each species. Doublestranded cDNA was synthesized from 5 μg of J. virginiana and 5 μg J.sabinoides pollen RNA as described in Example 3. The double-strandedcDNA was ligated to ethanol precipitated, self annealed, AT (SEQ ID NO:20) and AL (SEQ ID NO: 22) oligonucleotides for use in a modifiedAnchored PCR as described in Example 3. A number of Cry j I primers werethen used in combination with AP (SEQ ID NO: 21) in an attempt toisolate the Cry j I homologues from the two juniper species. Thesequences of AR (SEQ ID NO: 20), AL (SEQ ID NO: 22) and AP (SEQ ID NO:21) are given in Example 3. First, a primary PCR was carried out with100 pmol each of the oligonucleotides CP-10 (SEQ ID NO: 9) and AP (SEQID NO: 21). Three percent (3 μl) of this initial amplification was thenused in a secondary PCR with 100 pmoles each of CP-10 (SEQ ID NO: 9) andAPA (SEQ ID NO: 98), which has the sequence5′-GGGCTCGAGCTGCAGTTTTTTTTTTTTTTTG-3′, where nucleotides 1-15 representPst I and Xho I endonuclease restriction sites added for cloningpurposes, and nucleotide 33 can also be an A or C. A broad smear, withno discreet band, was revealed upon examination of the secondary PCRreactions on an EtBr-stained agarose gel. Attempts to clone Cry j Ihomologues from these PCR products were not successful. This approachwould have cloned a carboxyl portion of these genes. The degenerate Cryj I primers CP-1 (SEQ ID NO: 3), CP-4 (SEQ ID NO: 194), and CP-7 (SEQ IDNO: 6) as described in Example 3 were then each used in primary PCRswith AP (SEQ ID NO: 21) on the double stranded tinkered J. virginianaand J. sabinoides cDNAs. Various primer pair combinations were used insecondary PCRs as follows: CP-2 (SEQ ID NO: 4)/AP (SEQ ID NO: 21) andCP-4 (SEQ ID NO: 194)/AP (SEQ ID NO: 21) on the CP-1 (SEQ ID NO: 3)/AP(SEQ ID NO: 21) primary PCR amplification mixture, CP-2 (SEQ ID NO:4)/AP (SEQ ID NO: 21) and CP-5 (SEQ ID NO: 195)/AP (SEQ ID NO: 21) onthe CP-4 (SEQ ID NO: 194)/AP (SEQ ID NO: 21) primary PCR amplificationmixture, and CP-8 (SEQ ID NO: 7)/AP (SEQ ID NO: 21) on the CP-7 (SEQ IDNO: 6)/AP (SEQ ID NO: 21) primary PCR amplification mixture. Only thelast amplification, the CP-8 (SEQ ID NO: 7)/AP (SEQ ID NO: 21) secondaryPCR amplification, yielded a band upon examination on an EtBr-stainedminigel; the others gave smears that could not be cloned into pUC19.Both the J. virginiana and J. sabinoides secondary PCRs with CP-8 (SEQID NO: 7) and AP (SEQ ID NO: 21), described in Example 3, called JV21and JS17, respectively, resulted in amplified products that wereapproximately 200 base pairs long. The amplified DNA was recovered asdescribed in Example 3 and simultaneously digested with Xba I and Pst Iin a 50 μl reaction, precipitated to reduce the volume to 10 μl, andelectrophoresed through a preparative 2% GTG NuSeive low melt gel (FMC,Rockport, Me.). The appropriate sized DNA band was visualized by EtBrstaining, excised, and ligated into appropriately digested pUC19 forsequencing by the dideoxy chain termination method of Sanger et al.(supra) using a commercially available sequencing kit (Sequenase kit,U.S. Biochemicals, Cleveland, Ohio). Two JS17 clones (pUC19JS17d andpUC19JS17f) and one JV21 clone (pUC19JV21g) were sequenced, and found tocontain sequences homologous to the Cry j I nucleotide and deduced aminoacid sequences. The Cry j I homologues isolated from J. sabinoides andJ. virginiana RNA were designated Jun s I and Jun v I, respectively.

The Cry j I primers CP-9 (SEQ ID NO: 8) and CP-10 (SEQ ID NO: 9) shouldwork in primary and secondary PCRs, respectively, with AP to amplify thecarboxyl portion of the Jun s I and Jun v I cDNAs. The sequence of theseprimers are essentially identical to the sequences of Jun s I (SEQ IDNO: 94) and Jun v I (SEQ ID NO: 96), with the exception of 2 nucleotidesin CP-9 (SEQ ID NO: 8) (T instead of A in position 5 of CP-9 (SEQ ID NO:8), C instead of A in position 12), and 1 in CP-10 (SEQ ID NO: 9) (Cinstead of A in position 12 for Jun s I only). However, primary PCRswith CP-9 (SEQ ID NO: 8) and AP (SEQ ID NO: 21) and secondary PCRs withCP-10 (SEQ ID NO: 9) and AP (SEQ ID NO: 21) did not yield identifiableJun s I nor Jun v I product when viewed on an EtBr-stained agarose gel.Oligonucleotide J1 (SEQ ID NO: 99) was synthesized. J1 and allsubsequent oligonucleotides were synthesized on an ABI 394 DNA/RNAsynthesizer (Applied Biosystems, Foster City, Calif.). Primary PCRs werecarried out using AP (SEQ ID NO: 21) and J1 (SEQ ID NO: 99) with J.virginiana and J. sabinoides cDNAs. J1 has the sequence5′-CTAAAAATGGCTTCCCCA-3′, which corresponds to nucleotides 20-37 of Juns I (FIG. 16) (SEQ ID NO: 94) and nucleotides 30-47 of Jun v I (FIG. 17)(SEQ ID NO: 96). A secondary PCR amplification was performed on theprimary J1 (SEQ ID NO: 99)/AP (SEQ ID NO: 21) amplification of J.sabinoides cDNA using primers J2 (SEQ ID NO: 100) and AP (SEQ ID NO:21). J2 (SEQ ID NO: 100) has the sequence5′-CGGGAATTCTAGATGTGCAATTGTATCTTGTTA-3′, whereby nucleotides 1-13represent EcoR I and Xba I endonuclease restriction sites added forcloning purposes, and the remaining nucleotides correspond tonucleotides 65-84 in the Jun s I sequence (FIG. 16) (SEQ ID NO: 94). Thesecondary amplification from J. virginiana cDNA was performed with AP(SEQ ID NO: 21) and J3 (SEQ ID NO: 101), which has sequence5′-CGGGAATTCTAGATGTGCAATAGTATCTTGTTG-3′ whereby nucleotides 1-13represent EcoR I and Xba I endonuclease restriction sites added forcloning purposes and the remaining nucleotides correspond to nucleotides75-94 in the Jun v I sequence (FIG. 17) (SEQ ID NO: 96). No specificamplified product was observed in either secondary reaction. The primersdesignated ED (SEQ ID NO: 102) and EDT (SEQ ID NO: 103) were used at amolar ratio of 3:1 (ED:EDT) in conjunction with primers J1 (SEQ ID NO:99), J2 (SEQ ID NO: 100) and J3 (SEQ ID NO: 101), as described below.EDT (SEQ ID NO: 103) has the sequence5′-GGAATTCTCTAGACTGCAGGTTTTTTTTTTTTTTT-3′. The nucleotides 1 through 20of EDT (SEQ ID NO: 103) were added to the poly-T track to create EcoR I,Xba I, and Pst I endonuclease restriction sites for cloning purposes. ED(SEQ ID NO: 102) has the sequence 5′-GGAATTCTCTAGACTGCAGGT-3′,corresponding to nucleotides 1 to 21 of EDT (SEQ ID NO: 103). Theseoligonucleotides and their use have been previously described(Morgenstern et al. (1991) Proc. Natl. Acad Sci. USA 88:9690-9694). ED(SEQ ID NO: 102)/EDT (SEQ ID NO: 103) were used in primary PCRs witholigonucleotide J1 (SEQ ID NO: 99) for amplifications from J. sabinoidesand J. virginiana cDNAs, followed by secondary PCRs witholigonucleotides J2 (SEQ ID NO: 100) and APA (SEQ ID NO: 98) (for J.sabinoides) or J3 (SEQ ID NO: 101) and APA (SEQ ID NO: 98) (for J.virginiana). No specific product was identified from theseamplifications. A final set of PCRs with J1 (SEQ ID NO: 99), J2 (SEQ IDNO: 100), and J3 (SEQ ID NO: 101) was tried with oligonucleotide APA(SEQ ID NO: 98). APA was used in a primary PCR reaction with J1 (SEQ IDNO: 99) for J. sabinoides and J. virginiana, followed by secondaryamplifications with J2 (SEQ ID NO: 100) (for J. sabinoides) or J3 (SEQID NO: 101) (for J. virginiana) and APA (SEQ ID NO: 98). No specificproduct was identified from these amplifications. The degenerate primerCP-57 (SEQ ID NO: 104) was then synthesized. CP-57 (SEQ ID NO: 104) hasthe sequence 5′-GGCCTGCAGTTAACAGCGTTTGCAGAAGGTGCA-3′, wherein T atposition 10 can also be C, T at position 11 can also be C, A at position13 can also be G, G at position 16 can also be A, T, or C, G at position18 can also be T, T at position 19 can also be C, G at position 22 canalso be A, T or C, C at position 23 can also be G, A at position 24 canalso be C, G at position 25 can also be A, T, or C, A at position 27 canalso be G, G at position 28 can also be A, T, or C, G at position 29 canalso be C, T at position 30 can also be A, and G at position 31 can alsobe A. The nucleotides 1 through 9 of CP-57 (SEQ ID NO: 104) were addedto create a Pst I site for cloning purposes, the nucleotides 10 through12 are complementary to a stop codon and nucleotides 13 through 33 arecomplementary to coding strand sequence essentially encoding the aminoacids CysSerLeuSerLysArgCys (amino acids 347 through 353 of FIG. 4 b(SEQ ID NO: 2), corresponding to nucleotides 1167 through 1187 of FIG.4b (SEQ ID NO: 1)). This was used in a primary PCR with J1 (SEQ ID NO:99) on both J. sabinoides and J. virginiana double stranded TinkeredcDNA, followed by a secondary PCRs with CP-57 (SEQ ID NO: 104) and J2(SEQ ID NO: 100) for J. sabinoides and CP-57 (SEQ ID NO: 104) and J3(SEQ ID NO: 101) for J. virginiana. No PCR products were recovered.Three additional degenerate Cry j I oligonucleotides were synthesized.CP-62 (SEQ ID NO: 105) has sequence 5′-CCACTAAATATTATCCA-3′, wherein Aat position 3 can also be G, A at position 6 can also be G, T atposition 9 can also be A or G, and T at position 12 can also be A or G;this degenerate oligonucleotide sequence is complementary to the codingstrand sequence essentially encoding the amino acids TrpIleIlePheSerGly(amino acids 69 through 74 of FIG. 4 a (SEQ ID NO: 2), corresponding tonucleotides 333 through 349 of FIG. 4 a (SEQ ID NO: 1)). CP-63 (SEQ IDNO: 106) has sequence 5′-GCATCCCCATCTTGGGGATG-3′, wherein A at position3 can also be G, A at position 9 can also be G, T at position 12 canalso be C, G at position 15 can also be A, T, or C, and A at position 18can also be G; this degenerate oligonucleotide sequence is complementaryto the sequence capable of encoding the amino acidsHisProGlnAspGlyAspAla (amino acids 146-152 of FIG. 4 a (SEQ ID NO: 2),corresponding to nucleotides 564 to 583 of FIG. 4 a (SEQ ID NO: 1)).CP-64 (SEQ ID NO: 107) has the sequence 5′-GTCCATGGATCATAATTATT-3′,wherein T at position 6 can also be C, A at position 9 can also be G, Aat position 12 can also be G, A at position 15 can also be G, and A atposition 18 can also be G; this degenerate oligonucleotide sequence iscomplementary to the coding strand sequence capable of encoding theamino acids AsnAsnTyrAspProTrpThr (amino acids 243-249 of FIG. 4 b (SEQID NO: 2), corresponding to nucleotides 855 through 874 of FIG. 4 b (SEQID NO: 1)). AP was used in a primary PCR amplification with CP-62 (SEQID NO: 105), CP-63 (SEQ ID NO: 106), CP-64 (SEQ ID NO: 107) and CP-3(SEQ ID NO: 5) (described in Example 3) for both J. sabinoides and J.virginiana double-stranded Tinkered cDNA. A diagnostic PCR was performedon each primary reaction mixture. In this diagnostic PCR, 3% of theprimary reaction was amplified as described above using AP and CP-8. Forboth J. sabinoides and J. virginiana, the expected bands ofapproximately 200 base pairs were observed in diagnostic PCRs from theprimary PCR with AP (SEQ ID NO: 21) and CP-63 (SEQ ID NO: 106).

The degenerate primer CP-65 (SEQ ID NO: 108) was then synthesized. CP-65(SEQ ID NO: 108) has the sequence 5′-GCCCTGCAGTCCCCATCTTGGGGATGGAC-3′,wherein A at position 15 can also be G, T at position 18 can also be C,G at position 21 can also be G, A, T, or C, A at position 24 can also beG, and G at position 27 can also be A, T, or C. Nucleotides 1-9 of CP-65(SEQ ID NO: 108) were added to create a Pst I restriction site forcloning purposes, while the remaining degenerate oligonucleotidesequence is complementary to coding strand sequence essentially capableof encoding the amino acids ValHisProGlnAspGlyAsp (amino acids 145-151of FIG. 4 a (SEQ ID NO: 2), corresponding to nucleotides 561 through 580of FIG. 4 a (SEQ ID NO: 1)). AP was used in conjunction with CP-65 (SEQID NO: 108) in a secondary PCR of the primary AP (SEQ ID NO: 21)/CP-63(SEQ ID NO: 106) amplifications of J. sabinoides and J. virginianadescribed above. These reactions were designated JS42 for J. sabinoidesand JV46 for J. virginiana. Both secondary PCRs gave bands ofapproximately 600 base pairs when examined on 1% agarose minigelsstained with EtBr. The DNA from the JS42 and JV46 PCRs was recovered asdescribed in Example 3, simultaneously digested with Xba I and Pst I in15 μl reactions then electrophoresed through a preparative 2% GTGSeaPlaque low melt gel (FMC, Rockport, Me.). The appropriate sized DNAbands were visualized by EtBr staining, excised, and ligated intoappropriately digested pUC19 for sequencing by the dideoxy chaintermination method (Sanger et al., supra) using a commercially availablesequencing kit (Sequenase kit, U.S. Biochemicals, Cleveland, Ohio).Clones were sequenced using M13 forward and reverse primers (N.E.Biolabs, Beverly, Mass.) and internal sequencing primer J4 (SEQ ID NO:109) for both Jun s I and Jun v I. J4 (SEQ ID NO: 109) has the sequence5′-GCTCCACCATGGGAGGCA-3′ (nucleotides 177-194 of FIG. 16 (SEQ ID NO: 94)and nucleotides 187-204 of FIG. 17 (SEQ ID NO: 96)), which is the codingstrand sequence that essentially encodes amino acids SerSerThrMetGlyGly(amino acids 30 through 35 of Jun s I (SEQ ID NO: 94) and Jun v I (SEQID NO: 96) as shown in FIGS. 16 and 17, respectively).

The sequence of the Jun s I (SEQ ID NO: 94) clone designated pUC19JS42ewas found to be identical to that of clones pUC19JS17d and pUC19JS17f intheir regions of overlap, although they had different lengths in the 5′untranslated region. Clone pUC 19JS 17d had the longest 5′ untranslatedsequence. Nucleotides 1 through 141 of FIG. 16 (SEQ ID NO: 94)correspond to sequence of clone pUC19JS17d. Clone pUC19JS42e correspondsto nucleotides 1 through 538 of FIG. 16 (SEQ ID NO: 94).

The sequences of the Jun v I (SEQ ID NO: 96) clones designatedpUC19JV46a and pUC19JV46b were identical to the sequence of clonepUC19JV21g in their regions of overlap, with the exception thatnucleotide 83 of FIG. 17 (SEQ ID NO: 96) was A in clone pUC19JV21grather than the T shown. This nucleotide difference does not result in apredicted amino acid change. Clones pUC19JV46a, pUC19JV46b andpUC19JV21g correspond to nucleotides 1 through 548, 1 through 548 and 2through 151 of FIG. 17 (SEQ ID NO: 96), respectively.

The cDNAs encoding the remainder of the Jun s I (SEQ ID NO: 94) and Junv I (SEQ ID NO: 96) genes were cloned from the respective linkered cDNAsby using degenerate oligonucleotide CP66 (SEQ ID NO: 110), which has thesequence 5′-CATCCGCAAGATGGGGATGC-3′, wherein T at position 3 can also beC, G at position 6 can also be A, T, or C, A at position 9 can also beG, T at position 12 can also be C, and T at position 18 can also be C,and AP (SEQ ID NO: 21) in a primary PCR. The sequence of CP-66 (SEQ IDNO: 110) is complementary to that of CP-63 (SEQ ID NO: 106). A secondaryPCR was performed on 3% of the initial amplification mixture, with 100pmoles each of AP (SEQ ID NO: 21) and CP-67 (SEQ ID NO: 111), which hasthe sequence 5′-CGGGAATTCCCTCAAGATGGGGATGCGCT-3′, wherein A at position15 can also be G, T at position 18 can also be C, T at position 24 canalso be C, G at position 27 can also be A, T, or C, and C at position 28can be T. The nucleotide sequence 5′-CGGGAATTC-3′ of primer CP-67 (SEQID NO: 111) (bases 1 through 9 of SEQ ID NO: 111) were added to createan EcoR I restriction site for cloning purposes. The remainingoligonucleotide sequence essentially encodes amino acidsProGlnAspGlyAspAlaLeu (amino acids 147 through 153 of FIG. 4 a (SEQ IDNO: 2), corresponding to nucleotides 567 through 586 of FIG. 4 a (SEQ IDNO: 1)). The amplified DNA products, designated JS45 from the J.sabinoides amplification and JV49ii from the J. virginianaamplification, were purified as described in Example 3, digested withEcoR I and Xba I (JS45) or EcoR I and Asp718 I (JV49ii) andelectrophoresed through a preparative 1% low melt gel. The dominant DNAbands, which were approximately 650 bp in length, were excised andligated into pUC19 for sequencing. DNA was sequenced by the dideoxychain termination method (Sanger et al. supra) using a commerciallyavailable kit (sequenase kit, U.S. Biochemicals, Cleveland, Ohio).

Two clones, designated pUC19JS45a and pUC19JV49iia for Jun s I (SEQ IDNO: 94 (and Jun v I (SEQ ID NO: 96), respectively, were sequenced usingM13 forward and reverse primers (N.E. BioLabs, Beverly, MA) and internalsequencing primers J8 (SEQ ID NO: 112), J9 (SEQ ID NO: 113), and J12(SEQ ID NO: 114) for Jun s I, and J6 (SEQ ID NO: 115) and J11 (SEQ IDNO: 116) for Jun v I. J8(SEQ ID NO: 112) has the sequence5′-TAGGACATGATGATACAT-3′ (nucleotides 690-707 of FIG. 16 (SEQ ID NO:94)), which is the coding strand sequence essentially encoding aminoacids LeuGlyHisAspAspThr of Jun s I (SEQ ID NO: 282) (amino acids201-206 of FIG. 16 (SEQ ID NO: 95)). J9 (SEQ ID NO: 113) has thesequence 5′-GAGATCTACACGAGATGC-3′ (nucleotides 976-993 of FIG. 16 (SEQID NO: 94)) which is the coding strand sequence essentially encodingamino acids ArgSerThrArgAspAla of Jun s I ,(SEQ ID NO: 283) (amino acids297-302 of FIG. 16 (SEQ ID NO: 95)). J12 (SEQ ID NO: 114 has thesequence 5′-AAAACTATTCCCTTCACT-3′, wherein A at position 1 can also beG, and A at position 4 can also be T. This is the non-coding strandsequence that corresponds to coding strand sequence (nucleotides 875-892of FIG. 16 (SEQ ID NO: 94) encoding amino acids SerGluGlyAsnSerPhe (SEQID NO: 279) of Jun SI (amino acids 263-268 of FIG. 16 (SEQ ID NO: 95)).J6(SEQ ID NO: 115) has the sequence 5′-TAGGACATAGTGATTCAT-3′(nucleotides 700-717 of FIG. 17 (SEQ ID NO: 96)), which is the codingstrand sequence essentially encoding amino acids LeuGlyHisSerAspSer (SEQID NO: 280) of Jun v I (amino acids 201-206 of FIG. 17 (SEQ ID NO: 97)).J11 (SEQ ID NO: 116) has the sequence 5′-CCGGGATCCTTACAAATAACACATTAT-3′,where nucleotides 1-9 encode a BamH I restriction site for cloningpurposes and nucleotides 10-27 correspond to noncoding strand sequencecomplementary to nucleotides 1165-1182 of FIG. 17 (SEQ ID NO: 96) in the3′ untranslated region of Jun v I. The sequence of clone pUC19JS45acorresponds to nucleotides 527 through 1170 of FIG. 16 (SEQ ID NO: 94).The sequence of clone pUC29JV49iia corresponds to nucleotides 537through 1278 of FIG. 17 (SEQ ID NO: 96).

A full length clone of Jun s I was amplified using PCR. OligonucleotidesJ7 (SEQ ID NO. 117) and J10 (SEQ ID NO: 118) were used in a PCR reactionas above with J. sabinoides double stranded, linkered cDNA. J7 (SEQ IDNO: 117) has the sequence 5′-CCCGAATTCATGGCTTCCCCATGCTTA-3′, wherenucleotides 1-9 encode an EcoR I restriction site added for cloningpurposes and nucleotides 10-27 (corresponding to nucleotides 2643 ofFIG. 16 (SEQ ID NO: 94)) are the coding strand sequence that encodeamino acids MetAlaSerProCysLeu (SEQ ID NO: 281) of Jun s I (amino acids−21 to −16, FIG. 16 (SEQ ID NO: 95)). J10 (SEQ ID NO: 118) has thesequence 5′-CCGGGATCCCGTTTCATAAGCAAGATT-3′, where nucleotides 1-9 encodea BamH I restriction site added for cloning purposes and nucleotides10-27 are the non-coding strand sequence complementary to nucleotides1140-1157 from the 3′ untranslated region of Jun s I (FIG. 16 (SEQ IDNO: 94)). The PCR product, designated JS53ii, gave a band ofapproximately 1200 bp when examined on a 1% agarose minigel stained withEtBr. The DNA from the JS53ii PCR was recovered as described in Example3. After precipitation and washing with 70% EtOH, the DNA wassimultaneously digested with EcoR I and BamH I in a 15 μl reaction andelectrophoresed through a preparative 1% GTG SeaPlaque low melt gel(FMC, Rockport, ME). The appropriate sized DNA band was visualized byEtBr staining, excised, and ligated into appropriately digested pUC19for sequencing by the dideoxy chain termination method (Sanger et al.(1977) supra) using a commercially available sequencing kit (Sequenasekit, U.S. Biochemicals, Cleveland, OH). The resultant clone,pUC19JS53iib was partially sequenced using M13 forward and reverseprimers (N.E. Biolabs, Beverly, MA) and internal sequencing primer J4(SEQ ID NO: 109). The sequence of pUC19JS53iib that was determined wasidentical to that obtained from clones pUC19JS17d, pUC19JS42e, andpUC19JS45a. The nucleotide sequence of clone pUC19JS53iib corresponds tonucleotides 26 through 1157 of FIG. 16 (SEQ ID NO: 94).

The nucleotide and predicted amino acid sequences of Jun s I are shownin FIG. 16 (SEQ ID NO: 64 and 65). Jun s I has an open reading frame of1101 nucleotides, corresponding to nucleotides 26 through 1126 of FIG.16 (SEQ ID NO: 94), that can encode a protein of 367 amino acids.Nucleotides 1-25 and 1130-1170 of FIG. 16 (SEQ ID NO: 94) areuntranslated 5′ and 3′ regions, respectively. The initiating Met,encoded by nucleotides 26-28 of FIG. 16 (SEQ ID NO: 94), has beenidentified through the 89% identity of nucleotides 23 through 30(AAAAATGGC) of FIG. 16 (SEQ ID NO: 94) with the consensus sequenceencompassing the initiating Met in plants (AACAATGGC; Lutcke, supra).There is also an in-frame stop codon just 5′ of the codon encoding theinitiating Met. Amino acids −21 to −1 of FIG. 16 (SEQ ID NO: 95)correspond to a predicted leader sequence. The amino terminus of themature form of Jun s I was identified as amino acid 1 of FIG. 16 (SEQ IDNO: 95) through direct protein sequence analysis of purified Jun s I(Gross et al supra). The mature form of Jun s I, corresponding to aminoacids 1 through 346 of FIG. 16 (SEQ ID NO: 95), has a predictedmolecular weight of 37.7 kDa. Jun s I has three potential N-linkedglycosylation sites with the consensus sequence of Asn-Xxx-Ser/Thr.

The nucleic and predicted amino acid sequences of Jun v I are shown inFIG. 17 (SEQ ID NO: 96 and 97). Nucleotides 1-35 and 1130-1170 of SEQ IDNO: 96 are untranslated 5′ and 3′ regions, respectively. The initiatingMet, encoded by nucleotides 36-38 of FIG. 17 (SEQ ID NO: 96), wasidentified through the 89% identity of nucleotides 23 through 30(AAAAATGGC) of FIG. 17 (SEQ ID NO: 96) with the consensus sequenceencompassing the initiating Met in plants (AACAATGGC; Lutcke, supra).The nucleic acids of Jun s I (FIG. 16 (SEQ ID NO: 94)) and Jun v I (FIG.17 (SEQ ID NO: 96)) are identical in this region surrounding theinitiating Met. There are also 2 in-frame stop codons in the 5′untranslated region of FIG. 17 (SEQ ID NO: 96). Jun v I has an openreading frame of 1,110 nucleotides, corresponding to nucleotides 36through 1145 of FIG. 17 (SEQ ID NO: 96), that can encode a protein of370 amino acids. Nucleotides 1146-1148 of FIG. 17 (SEQ ID NO: 96) encodea stop codon. Amino acids −21 to −1 of Jun v I (FIG. 17 (SEQ ID NO: 97))correspond to a predicted leader sequence. The amino terminus of themature form of Jun v I was identified as amino acid 1 of FIG. 17 (SEQ IDNO: 97) by comparison with the sequences of Cry j I (FIG. 4 a) (SEQ IDNO: 2) and Jun s I (FIG. 16) (SEQ ID NO: 95). The mature form of Jun vI, corresponding to amino acids 1 through 349 of FIG. 17 (SEQ ID NO: 97)has a predicted molecular weight of 38.0 kDa. Jun v I has four potentialN-linked glycosylation sites with the consensus sequence ofAsn-Xxx-Ser/Thr.

As shown in Table 1, the amino acid sequences of the mature forms of Juns I and Jun v I are 80.9% homologous (75.4% identity and 5.5%similarity) with each other. The amino acid sequences of the matureforms of Jun s I and Cry j I are 87% homologous (80.1% identity, 6.9%similarity) and the sequences of the mature forms of Jun v I and Cry j Iare 80.5% homologous (72.5% identity, 8% similarity). The homologiesbetween Cry j I peptide sequences identified in Example 6 as containingT cell epitopes and the corresponding Jun s I and Jun v I sequences arealso very high. For example, peptide CJ1-22 (SEQ ID NO: 47) (FIG. 13),corresponding to amino acids 211-230 of Cry j I (FIG. 4 b) (SEQ ID NO:2), contains a major T cell epitope (FIG. 14). CJ1-22 (SEQ ID NO: 47)has 95% identity (19/20 identical amino acids) and 85% homology (16/20identical amino acids, 1/20 similar amino acid) with the correspondingregions of Jun s I (SEQ ID NO: 95) and Jun v I (SEQ ID NO: 97),respectively (see Table I). This high degree of sequence homologysuggests that an immunotherapy effective in treating allergic diseasecaused by Cry j I may also be effective in treating allergic diseasescaused by Cry j I homologues. All nucleic and amino acid analyses wereperformed using software contained in PCGENE (Intelligenetics, MountainView, Calif.).

TABLE I Protein/Peptide Total Comparisons Identity Similarity HomologyJun s I vs. Jun v I 75.4% 5.5% 80.9% Jun s I vs. Cry j I 80.1% 6.9%87.9% Jun v I vs. Cry j I 72.5% 8.0% 80.5% CJ1-22 vs. Jun s I₂₁₁₋₂₃₀95.0% 0.0% 95.0% CJ1-22 vs. Jun v I₂₁₁₋₂₃₀ 80.0% 5.0% 85.0% Native Jun sI or Jun v I can also be biochemically purified using known techniquesor purified by other means to a high degree of purity by amino acidsequencing of the purified native product and comparing the sequence ofthe purified native product to the amino acid sequence of Jun s I or Junv I provided herein.

EXAMPLE 10

Northern blot analysis of C. japonica, J. sabinoides, J. virginiana andC. arizonica RNA.

A Northern blot analysis was performed on RNA isolated from C. japonica,J. sabinoides and J. virginiana pollens. RNA from C. japonica pollenscollected in both the United States (Example 3) and Japan (Example 4)were examined. Using essentially the method of Sambrook, supra, 15 μg ofeach RNA were run on a 1.2% agarose gel containing 38% formaldehyde and1×MOPS (20×=0.4M MOPS, 0.02M EDTA, 0.1 M NaOAc, pH 7.0) solution. TheRNA samples (first precipitated with 1/10 volume sodium acetate, 2volumes ethanol to reduce volume and resuspended in 5.5 μl dH2O) wererun with 10 μl formaldehyde/formamide buffer containing loading dyeswith 15.5% formaldehyde, 42% formamide, and 1.3×MOPS solution, finalconcentration. The samples were transferred to Genescreen Plus (NENResearch Products, Boston, Mass.) by capillary transfer in 10×SSC(20×=3M NaCl, 0.3M Sodium Citrate), after which the membrane was baked 2hr. at 80° C. and UV irradiated for 3 minutes. Prehybridization of themembrane was at 60° C. for 1 hour in 4 ml 0.5M NaPO4 (pH 7.2), 1 mMEDTA, 1% BSA, and 7% SDS. The antisense probe was synthesized byasymmetric PCR (McCabe, P. C., in: PCR Protocols. A Guide to Methods andApplications, Innis, M., et al., eds. Academic Press, Boston, (1990), pp76-83) on the JC91a amplification in low melt agarose (described inExample 3), where 2 μl DNA is amplified with 2 μl dNTP mix (0.167 mMdATP, 0.167mM dTTP, 0.167mM dGTP, and 0.033mM dCTP), 2 μl 10×buffer, 10μl ³²P-dCTP (100 μCi; Amersham, Arlington Heights, Ill.), 1 μl (100pmoles) antisense primer CP-17 (SEQ ID NO: 14), 0.5 μl Taq polymerase,and dH₂O to 20 μl; the 10×PCR buffer, dNTPs and Taq polymerase were fromPerkin Elmer Cetus (Norwalk, Conn.). Amplification consisted of 30rounds of denaturation at 94° C. for 45 sec, annealing of primer to thetemplate at 60° C. for 45 sec, and chain elongation at 72° C. for 1 min.The reaction was stopped by addition of 100 μl TE, and the proberecovered over a 3 cc G-50 spin column (2 ml G-50 Sephadex [Pharmacia,Uppsala, Sweden] in a 3 cc syringe plugged with glass wool, equilibratedwith TE) and counted on a 1500 TriCarb Liquid Scintillation Counter(Packard, Downers Grove, Ill.). The probe was added to theprehybridizing buffer at 10⁶ cpm/ml and hybridization was carried out at60° C. for 16 hrs. The blot was washed in high stringency conditions:3×1 min at 65° C. with 0.2×SSC/1% SDS, followed by wrapping in plasticwrap and exposure to film at −80° C. A seven hour exposure of thisNorthern blot revealed a single thick band at approximately 1.2 kb forC. japonica (United States) (FIG. 19 a, lane 1), C. japonica (Japan)(FIG. 19 a, lane 2), J. sabinoides (FIG. 19 a, lane 3) and J. virginiana(FIG. 19 a, lane 4) RNAs. This band is the expected size for Cry j I,Jun s I and Jun v I as predicted by PCR analysis of the cDNA. Thedifferent band intensities in each lane may reflect differences in theamount of RNA loaded on the gel. The position of 1.6 and 1.0 kbmolecular weight standards are shown on the FIGS. 19 a and 19 b.

RNA isolated from J. sabinoides and C. arizonica were analyzed in aseparate Northern blot. Five μg of total RNA from J. sabinoides and 5 μgof total RNA from C. arizonica were probed as described. The 1.2 kb bandwas observed in this blot for both J. sabinoides (FIG. 19 b, lane 1) andC. arizonica (FIG. 19 b, lane 2), indicating that C. arizonica has a Cryj I homologue. Other, related, trees are also expected to have a Cry j Ihomologue.

EXAMPLE 11

Japanese Cedar Pollen Allergic Patient T Cell Studies with Cry j I—thePrimary Cedar Pollen Antigen.

Synthesis of Peptides

Japanese cedar pollen Cry j I peptides were synthesized using standardFmoc/tBoc synthetic chemistry and purified by Reverse Phase HPLC. FIG.20 shows Cry j I peptides used in these studies. The peptide names areconsistent throughout.

T Cell Responses to Cedar Pollen Antigen Peptides

Peripheral blood mononuclear cells (PBMC) were purified by lymphocyteseparation medium (LSM) centrifugation of 60 ml of heparinized bloodfrom Japanese cedar pollen-allergic patients who exhibited clinicalsymptoms of seasonal rhinitis and were MAST and/or skin test positivefor Japanese cedar pollen. Long term T cell lines were established bystimulation of 2×10⁶ PBL/ml in bulk cultures of complete medium(RPMI-1640, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, 5×10⁻⁵M2-mercaptoethanol, and 10 mM HEPES supplemented with 5% heat inactivatedhuman AB serum) with 20 μg/ml of partially purified native Cry j I (75%purity containing three bands similar to the three bands in FIG. 2) for6 days at 37° C. in a humidified 5% CO₂ incubator to select for Cry j Ireactive T cells. This amount of priming antigen was determined to beoptimal for the activation of T cells from most cedar pollen allergicpatients. Viable cells were purified by LSM centrifugation and culturedin complete medium supplemented with 5 units recombinant human IL-2/mland 5 units recombinant human IL4/ml for up to three weeks until thecells no longer responded to lymphokines and were considered “rested”.The ability of the T cells to proliferate to selected Cry j I peptides,partially purified Cry j I, affinity purified Cry j I, or positive (PHA)controls or negative controls (medium only) was then assessed. Forassay, 2×10⁴ rested cells were restimulated in the presence of 2×10⁴autologous Epstein-Barr virus (EBV)-transformed B cells (prepared asdescribed below) (gamma-irradiated with 25,000 RADS) with 2-50 μg/ml ofrCry j I, purified native Cry j I in a volume of 200 μl complete mediumin duplicate or triplicate wells in 96-well round bottom plates for 2-4days. The optimal incubation was found to be 3 days. Each well thenreceived 1 μCi tritiated thymidine for 16-20 hours. The countsincorporated were collected onto glass fiber filter mats and processedfor liquid scintillation counting. Titrations using T cells from oneindividual were conducted which showed the effect of varying antigendose in assays with purified native Cry j I and several the peptidessynthesized as described above. The titrations were used to optimize thedose of peptides in T cell assays.

The maximum response in a titration of each peptide is expressed as thestimulation index (S.I.). The S.I. is the counts per minute (CPM)incorporated by cells in response to peptide, divided by the CPMincorporated by cells in medium only. An S.I. value equal to or greaterthan 2 times the background level is considered “positive” and indicatesthat the peptide contains a T cell epitope. The positive results wereused in calculating mean stimulation indices for each peptide for theindividual patient tested.

The above procedure was followed with 39 patients. Individual patientresults were used in calculating the mean S.I. for each peptide if thepatient responded to the Cry j I protein at an S.I. of 2.0 or greaterand the patient responded to at least one peptide derived from Cry j Iat an S.I. of 2.0 or greater. A summary of positive experiments fromthirty-nine (n=39) patients is shown in FIG. 21. The bars represent thepositivity index. Above each bar is the percent of positive responseswith an S.I. of at least two to the peptide or protein in the group ofpatients tested. In parenthesis above each bar are the mean stimulationindices for each peptide or protein for the group of patients tested.All but one of the thirty-nine T cell lines responded to purified nativeCry j I. However, the one T cell line which did not respond to purifiednative Cry j I did respond to peptides derived from Cry j I. This panelof Japanese cedar allergic patients responded to peptides: CJ1-42.5 (SEQID NO: 119), CJ1-42.8 (SEQ ID NO: 120), CJ1-43.26 (SEQ ID NO: 121),CJ1-43.27 (SEQ ID NO: 122), CJ1-43.30 (SEQ ID NO: 123), CJ1-43.31 (SEQID NO: 124), CJ1-43.32 (SEQ ID NO: 125), CJ1-43.35 (SEQ ID NO: 126),CJ1-43.36 (SEQ ID NO: 127), CJ1-43.39 (SEQ ID NO: 128), CJ1-24.5 (SEQ IDNO: 129), CJ1-44.5 (SEQ ID NO: 130), CJ1-44.6 (SEQ ID NO: 131), CJ1-44.8(SEQ ID NO: 132) all as shown in FIG. 20, indicating that these peptidescontain T cell epitopes. Preparation of (EBV)-transformed B Cells forUse as Antigen Presenting Cells was described in Example 6, supra.

EXAMPLE 12

Cry j I Peptide Screen.

To analyze IgE reactivity to the selected peptides discussed in example11 and shown in FIG. 20, a direct ELISA format was used. ELISA wellswere coated with the selected peptides derived from Cry j I and thenassayed for IgE binding. FIGS. 22 and 23 are graphs of these bindingresults using two different pools of Cry j allergic patient plasma.Patient plasma pool A (denoted PHP-A) (FIG. 22) was formulated by mixingequal volumes of plasma from 22 patients that were all shown to bepositive for direct IgE binding to native purified Cry j I by ELISA. Thesecond pool (PHP-D) (FIG. 23) was formulated by the combination of equalplasma volumes from 8 patients that had IgE binding by direct ELISA toboth native and denatured purified Cry j I. This pool was generated toincrease the chance of detecting reactivity towards peptides. Both poolsin this assay set show direct binding to the native purified Cry j I,FIG. 22 and FIG. 23. There was no detectable IgE binding reactivity toany of the peptides at any of the plasma concentrations used. To controlfor the presence of peptide coating the wells, mouse polyclonal antiserawas generated to the peptides. These antisera were then used in directELISA binding to demonstrate that the peptides were coating the wells.The results of these assays are shown in FIG. 24, and indicate thatpeptides were coating the wells.

In addition, 20 allergic patients which demonstrated IgE binding to Cryj I were examined for IgE reactivity to peptides CJ1-24.5, CJ1-43.39,and CJ1-44.8 using essentially the same protocol described above. Nopatient showed IgE binding to peptides CJ1-24.5, CJ1-43.39, andCJ1-44.8, or to the controls of patient plasma on oncoated blocked wells(gelatin) or to an irrelevant peptide (data not shown).

EXAMPLE 13

Purification of Native Japanese Cedar Pollen Allergen (Cry j II)

The following purification of native Cry j II from Japanese cedar pollenwas modified from previously published reports (Yasueda et al, J.Allergy Clin. Immunol. 71:77 (1983); Sukaguchi et al., Allergy, 45:309(1990)).

100 g of Japanese cedar pollen obtained from Japan (Hollister-Stier,Spokane, Wash.) was defatted in 1 L diethyl ether three times, thepollen was collected after filtration and the ether was dried off in avacuum.

The defatted pollen was extracted at 4° C. overnight in 2 L extractionbuffer containing 50 mM tris-HCl, pH 7.8, 0.2 M NaCl and proteaseinhibitors in final concentrations: soybean trypsin inhibitor (2 μg/mL),leupeptin (1 μg/mL), pepstatin A (1 μg/mL) and phenyl methyl sulfonylfluoride (0.17 mg/mL). The insoluble material was re-extrated with 1.2 Lextraction buffer at 4° C. overnight and both extracts were combinedtogether and depigmented by batch absorption with Whatman DE-52 (200 gdry weight) equilibrated with the extraction buffer.

The depigmented material was then fractionated by ammonium sulfateprecipitation at 80% saturation (4° C.), which removed much of the lowermolecular weight material. The resulting pellet was resuspended in 0.4 Lof 50 mM Na-acetate, pH 5.0 containing protease inhibitors and wasdialyzed extensively against the same buffer.

The sample was further subjected to purification by either one of thetwo methods described below.

Method A

The sample was applied to a 100 mL DEAE cellulose column (Whatman DE-52)equilibrated at 4° C. with 50 mM Na-acetate, pH 5.0 with proteaseinhibitors. The unbound material (basic proteins) from the DEAEcellulose column was then applied to a 50 ml cation exchange column(Whatman CM-52) which was equilibrated with 10 mM Na-acetate, pH 5.0 at4° C. with protease inhibitors. A linear gradient of 0-0.3 M NaCl wasused to elute the proteins. The early fractions were enriched in Cry j Iwhereas the later fractions were enriched in Cry j II. Fractionscontaining Cry j II were pooled and next applied to an 1 mL Mono S HR5/5 column (Pharmacia, Piscataway, N.J.) in 10 mM Na-acetate, pH 5.0,and proteins were eluted with a linear gradient of NaCl at roomtemperature. Residual Cry j I was eluted at −0.2 M NaCl and Cry j II waseluted between 0.3 to 0.4 M NaCl. The Cry j II peak was pooled andconcentrated to twofold by lyophilization and subjected to gelfiltration chromatography.

The sample was applied to FPLC Superdex 75 16/60 column (Pharmacia,Piscataway, N.J.) in 10 mM acetate buffer, pH 5.0 and 0.15 M NaCl at aflow rate of 30 ml/min. at room temperature. Purified Cry j II wasrecovered in the 35-30 kD region. Cry j II migrated as two broad bandslower than Cry j I under non-reducing conditions (FIG. 25 a) but bothbands shifted upward and migrated as Cry j I under reducing condition(FIG. 25 b) when analyzed by silver-stained SDS-PAGE. This highlypurified Cry j II still contained a small amount (−5%) of Cry j I asdetected by Western blot using MAb CBF2, which has been shown to bind toCry j I and by N-terminal protein sequencing. This Cry j II preparationwas used to generate primary protein sequence of Cry j II as describedbelow.

Method B

The dialyzed sample from the ammonium sulfate precipitation was appliedat 1 ml/min to an 5.0 ml Q-Sepharose Econapac anion exchange cartridge(BioRad, Richmond, Calif.) equilibrated with 50 mM Na-acetate, pH 5.0with protease inhibitors at 4° C. Elution was performed with the abovebuffer containing 0.5 M NaCl. The basic unbound material was thenapplied to a 5.0 ml CM-Sepharose Econopac cation exchange cartridge(BioRad, Richmond, Calif.) equilibrated in 50 mM sodium acetate pH 5.0with protease inhibitors. Basic proteins were eluted with a lineargradient up to 0.1 M sodium phosphate pH 7.0, 0.3 M NaCl at 1 ml/min at4° C. A Cry j II-enriched peak was collected late in the gradient andfurther purified by gel filtration chromatography.

FPLC gel filtration was performed using a 320 mL Superdex 75 26/60(Pharmacia, Piscataway, N.J.) column at 0.5 ml/min in 20 mM sodiumacetate, pH 5.0, in the presence of 0.15 M NaCl. The major peakcontaining mostly Cry j II eluted between 160 and 190 ml. ContaminatingCry j I was next removed by FPLC using a 1.0 ml Mono 5 S 5/5 (Pharmacia,Piscataway, N.J.) cation exchange column equilibrated with 10 mM sodiumacetate pH 5.0. A stepwise gradient of 0-1 M NaCl was utilized byholding isocratically at 0.2 M, 0.3 M, 0.4 M and 1 M salt concentration.

Multiple peaks (up to nine peaks) were obtained (FIG. 26) and analyzedby silver stained SDS-PAGE under reducing conditions (FIG. 27). Cry j Iwith a reported pI of 8.6-8.9 (Yasueda et al, J. Allergy Clin. Immunol.,17 (1983)), eluted in the earlier peaks and displayed a molecular weightof about 40 kD. Cry j II was purified to homogeneity as two bands (FIG.27) and eluted in the later multiple peaks, suggesting the existence ofisoforms. ELISA analysis using the mouse monoclonal 8B11 IgG antibodywhich was raised against biochemically purified Cry j I confirmed theabsence of Cry j I in these purified Cry j II preparation. This purifiedCry j II was used in the human IgE reactivity studies (Example 18).

Physical Properties of Cry j II

The physiochemical properties of Cry j II were studied and summarized asbelow. Under non-reducing SDS-PAGE conditions Cry j II consists of twobands with molecular weights ranged 34000-32000. The molecular weightsof both bands are shifted higher to about 38-36 kD under reducingconditions (FIG. 25 b). This shift in SDS-polyacrylamide gel has alsobeen observed by others (Sakaguchi et al, Allergy 45:309-312 (1990)).These results suggest that intra-disulfide bonds are probably present inthe protein, and it is supported by the present findings that cloned Cryj II contains 20 cysteines deduced from the nucleotide sequence (Example15). The pI of Cry j II estimated from IEF gel is about 10. The purifiedCry j II binds human IgE of some allergic patients.

The two molecular weight bands of Cry j II were separated on a 12%SDS-polyacrylamide gel and was then electroblotted onto PVDF membrane(Applied Biosystems, Foster City, Calif.). The blot was stained withcoomassie brilliant blue and was cut and subjected to N-terminal aminoacid sequencing. (Example 14). The results showed that the upper andlower molecular weight bands had identical N-terminal sequences exceptthe lower molecular weight band missed the first five amino acids. Theestimated molecular weight of the upper band based on the cDNA sequenceis about 52,000, which is significantly higher than the molecular weightestimated from SDS-polyacrylamide gel either in the presence or absenceof reducing reagent. It is also higher than that obtained from gelfiltration and preliminary mass spectroscopy analysis. These are severalpossibilities to account for this difference. One possibility is thatCry j II protein is processed. It is probable that the N-terminal andC-terminal of the protein are cleaved. It is not clear at the presenttime whether this processing occurs in the cell or due to proteolysisduring purification even though four different protease inhibitors wereadded in most of the purification steps. Nevertheless, the twoN-terminal sequences obtained from the purified Cry j II (Example 14)also contained the N-terminal sequence (10 amino acid) published bySakaguchi et al (Allergy, 45:309-312(1990)) suggesting that theN-terminal of Cry j II is probably hydrolyzed. Since Sakaguchi et al.(supra), did not use any protease inhibitors in their purification, ahigher degree of hydrolysis might have occurred. This could explain whythe N-terminal amino acid sequence that Sakaguchi et al. obtained wasdownstream of the N-terminal sequences as discussed in Example 14.

Another approach which may be used to purify native Cry j II orrecombinant Cry j II is immunoaffinity chromatography. This techniqueprovides a very selective protein purification due to the specificity ofthe interaction between monoclonal antibodies and antigen. Murinepolyclonal and monoclonal antibodies are generated against purified Cryj II. These antibodies are used for purification, characterization,analysis and diagnosis of the allergen Cry j II.

EXAMPLE 14

Protein Sequencing of Purified Cry j II

Cry j II protein was isolated as in Example 1. The doublet band shown onSDS-PAGE (FIG. 25 a) was electroblotted onto ProBlott (AppliedBiosystems, Foster City, Calif.). Sequencing was performed with theBeckman/Porton Microsequencer (model LF3000, Beckman Instruments,Carlsbad, Calif.), a Programmable Solvent Module (Beckman System GoldModel 126, Beckman Instuments, Carlsbad, Calif.) and a Diode ArrayDetector Module for PTH-amino acid detection (Beckman System Gold Model168, Beckman Instruments, Carlsbad, Calif.) following manufacturersspecifications.

A single N-terminal sequence analysis of the upper doublet band andmultiple N-terminal sequence analyses of the lower doublet band showedthat both bands contained two N-termini, designated “long” and “short”.The lower doublet band contained approximately 3.3 picomoles of the longform and 8.3 picomoles of the short form. This difference in yields wassufficient to make sequence assignments according to the quantitation ateach sequencer cycle. The upper doublet band contained approximately 8.3picomoles of both sequences. The revealed long sequence wasNH₂—RKVEHSRHDAINIFNVEKYGAVGDGKHDCTEAFSTAW(Q) ( ) ( ) ( ) KNP ( ) —COOH(SEQ ID NO: 136), where (Q) indicates a tentative identification ofglutamine at position 38 and 0 indicated unknown residues at positions39-41 and 45. The revealed “short” sequence wasNH₂—SRHDAINIFNVEKYGAVGDGKHDCTEAFSTAWS—COOH (SEQ ID NO: 137). Thus thelong Cry j II sequence had five additional amino terminal residues thanthe short form and the sequence of the short form exactly matched thatof the long form. In addition, both the long and short forms of Cry j IIcontained the ten amino acids, NH₂-AINIFNVEKY—COOH (SEQ ID NO: 138),previously described for Cry j II (Sakaguchi et al. 1990, supra). Thepreviously published 10 amino acids (Sakaguchi et al. 1990, supra)correspond to amino acids ten through 19 of the long form describedabove (SEQ ID NO: 136).

EXAMPLE 15

Extraction of RNA From Japanese Cedar Pollen and Staminate Cones andCloning of Cry j II

Fresh pollen and staminate cone samples, collected from a singleCryptomeria japonica (Japanese Cedar) tree at the Arnold Arboretum(Boston, Mass.), were frozen immediately on dry ice. RNA was preparedfrom 500 mg of each sample, essentially as described by Frankis andMascarhenas (1980) Ann. Bot. 45: 595-599. The samples were ground bymortar and pestle on dry ice and suspended in 5 ml of 50 mM Tris pH 9.0with 0.2 M NaCl, 1 mM EDTA, 0.1% SDS that had been treated overnightwith 0. 1% diethyl pyrocarbonate (DEPC). After five extractions withphenol/chlorofom/isoamyl alcohol (mixed 25:24:1), the RNA wasprecipitated from the aqueous phase with 0.1 volume 3M sodium acetateand 2 volumes ethanol. The pellets were recovered by centrifugation,resuspended in 2 ml dH₂O and heated to 65° C. for 5 minutes. Two ml 4Mlithium chloride was added to the preparation and the RNA wasprecipitated overnight at 0° C. The RNA pellets were recovered bycentrifugation, resuspended in 1 ml dH₂O, and again precipitated with 3Msodium acetate and ethanol on dry ice for one hour. The final pellet waswashed with 70% ethanol, air dried and resuspended in 100 μlDEPC-treated dH₂O and stored at −80° C.

Double stranded cDNA was synthesized from 4 μg pollen RNA or 8 μgflowerhead RNA using a commercially available kit (cDNA Synthesis Systemkit, BRL, Gaithersburg, Md.). The double-stranded cDNA was phenolextracted, ethanol precipitated, blunted with T4 DNA polymerase(Promega, Madison, Wis.), and then ligated to ethanol precipitated, selfannealed, AT and AL oligonucleotides for use in a modified Anchored PCRreaction, according to the method of Rafnar et al. (1990) J. Biol. Chem.266: 1229-1236 ; Frohman et al. (1990) Proc. Natl. Acad. Sci. USA 85:8998-9002; and Roux et al. (1990) BioTech. 8: 48-57. Oligonucleotide AThas the sequence 5′-GGGTCTAGAGGTACCG-TCCGTCCGATCGATCATT-3′ (SEQ ID NO:20) (Rafnar et al. supra). Oligonucleotide AL has the sequence5′-AATGATCGATGCT (SEQ ID NO: 22) (Rafnar et al. supra).

The first attempts at amplifying the amino terminus of Cry j II from thelinkered cDNA (2 μl of a 20 μl reaction) was made using the degenerateoligonucleotide CP-11 and oligonucleotide AP. CP-11 has the sequence5′-ATACTTCTClACGTTGAA-3′ (SEQ ID NO: 142), wherein A at positon 1 can beG, C at position 4 can be T, C at position 7 can be T, I at position 10is inosine to reduce degeneracy (Knoth et al. (1988) Nucleic Acids Res.16: 10932), G at position 13 can be A, and G at position 16 can be A).AP, which has the sequence 5′-GGGTCTAGAGGTA-CCGTCCG-3′ (SEQ ID NO: 21),corresponds to nucleotides 1 through 20 of the oligonucleotide AT (SEQID NO: 20). CP-11 (SEQ ID NO: 142) is the degenerate oligonucleotidesequence that is complementary to the coding strand sequencesubstantially encoding amino acids PheAsnValGluLysTyr (SEQ ID NO:143)(amino acids 59 to 64 of (SEQ ID NO: 134), (FIG. 28) whichcorrespond to the carboxy terminus of the previously published Cry j IIsequence (Sakaguchi et al., supra) shown in FIG. 28. Alloligonucleotides were synthesized by Research Genetics Inc., Huntsville,Ala.

Polymerase chain reactions (PCR) were carried out using a commerciallyavailable kit (GeneAmp DNA Amplification kit, Perkin Elmer Cetus,Norwalk, Conn.) whereby 10 μl 10× buffer containing dNTPs was mixed with100 pmoles of each oligonucleotide, cDNA (3-5 μl of a 20 μl first strandcDNA reaction mix), 0.5 μl Amplitaq DNA polymerase, and distilled waterto 100 μl.

The samples were amplified with a programmable thermal controller (MJResearch, Inc., Cambridge, Mass.). The first 5 rounds of amplificationconsisted of denaturation at 94° C. for 1 min, annealing of primers tothe template at 45° C. for 1 min, and chain elongation at 72° C. for 1min. The final 20 rounds of amplification consisted of denaturation asabove, annealing at 55° C. for 1 min, and elongation as above. Theprimary PCR reaction was carried out with 100 pmol each of theoligonucleotides AP (SEQ ID NO: 21) and CP-11 (SEQ ID NO: 142). Fivepercent (5 μl) of this initial amplification was then used in asecondary amplification with 100 pmoles each of AP (SEQ ID NO: 21) andCP-12. CP-12 has the sequence 5′-CCTGCAGTACTTCT-CIACGTTGAAIAT-3′ (SEQ IDNO: 144), wherein C at position 10 can be T, C at position 13 can be T,I at positions 16 and 25 are inosines to reduce degeneracy as above, Gat position 19 can be A, and G at position 22 can be A. The sequence5′-CCTGCAG-3′ (SEQ ID NO: 145) (bases 1 through 7 of CP-12) (SEQ ID NO:144) represents a Pst I site added for cloning purposes; the remainingdegenerate oligonucleotide sequence is complementary to the codingstrand sequence that substantially encodes the amino acidsIlePheAsnValGluLysT (SEQ ID NO: 146) (amino acids 58-64 of SEQ ID NO:134; FIG. 28). Amplified DNA was recovered by sequential chloroform,phenol, and chloroform extractions, followed by precipitation on dry icewith 0.5 volumes of 7.5M ammonium acetate and 1.5 volumes ofisopropanol. After precipitation and washing with 70% ethanol, the DNAwas simultaneously digested with Xba I and Pst I in a 50 μl reaction,precipitated to reduce the volume to 10 μl, and electrophoresed througha preparative 2% GTG NuSeive low melt gel (FMC, Rockport, Me.). Theappropriate sized DNA area was visualized by ethidium bromide (EtBr)staining, excised, and ligated into appropriately digested pUC19 forsequencing by the dideoxy chain termination method of Sanger et al.(1977) Proc. Natl. Acad. Sci. USA 74: 5463-5476) using a commerciallyavailable sequencing kit (Sequenase kit, U.S. Biochemicals, Cleveland,Ohio). All resultant clones were sequenced, and none were found tocontain Cry j II sequence. An alternate 2° PCR reaction was performedwith AP (SEQ ID NO: 21) and the nested oligonucleotide CP-21. CP-21 hasthe sequence 5′-CCTGCAGTACTTCTCIACGTTGAAGAT-3′ (SEQ ID NO: 147) whereinC at position 10 can be T, C at position 13 can be T, I at position 16is inosine to reduce degeneracy as above, G at position 19 can be A, Gat position 22 can be A, and G at position 25 can be A or T. Thesequence 5′-CCTGCAG-3′ (SEQ ID NO: 145) (bases 1 through 7 of CP-21)(SEQ ID NO: 147) represent a Pst I site added for cloning purposes; theremaining degenerate oligonucleotide sequence is the non-coding strandsequence corresponding to coding strand sequence substantially encodingamino acids IlePheAsnValGluLysTyr (SEQ ID NO: 146) (amino acids 58 to 64of SEQ ID NO: 134; FIG. 28).

A primary PCR was also performed on double-stranded, Tinkered cDNA usingCP-23D (SEQ ID NO: 148) and AP (SEQ ID NO: 21), as above, to attempt toamplify the 3′ end of the Cry j II cDNA. A secondary PCR was performedusing 5% of the primary reaction, using CP-24D (SEQ ID NO: 150) and AP(SEQ ID NO: 21). CP-23D (sequence 5′-GCIATTAATATTTTAA-3′,(SEQ ID NO:148) wherein the T at position 6 can be C or A, T at position 9 can beC, T at position 12 can be C or A, and T at position 15 can be C ) isthe coding strand sequence substantially encoding amino acidsAlalleAsnIlePheAsn (SEQ ID NO: 149) (amino acids 55 to 60 of SEQ ID NO:134; FIG. 28); CP-24D (sequence 5′-GGAATTCCGCIATTAATATTTTAATGT-3′ (SEQID NO: 150), wherein the T at position 14 can be C or A, T at position17 can be C, T at position 20 can be C or A, T at position 23 can be C,and T at position 26 can be C ) contains the sequence 5′-GGAATTCC-3′(SEQ ID NO: 151) (bases I through 8 of CP-24D (SEQ ID NO: 150)), whichrepresents an Eco RI site added for cloning purposes. The remainingdegenerate oligonucleotide sequence of CP-24D (SEQ ID NO: 150)substantially encodes amino acids AlalleAsnIlePheAsnVal (SEQ ID NO: 152)(amino acids 55 to 61 of SEQ ID NO: 134; FIG. 28). Again, multipleclones were sequenced, none of which could be identified as Cry j II,and this approach was not pursued further.

Upon the characterization of novel Cry j II protein sequence datadescribed in Example 14, new degenerate oligonucleotides for cloning Cryj II were designed and synthesized. All oligonucleotides mentionedhereafter were synthesized on an ABI 394 DNA/RNA Synthesizer (AppliedBiosystems, Foster City, Calif.), and purified on NAP-10 columns(Pharmacia, Uppsala, Sweden) as per the manufacturers' instructions.Degenerate oligonucleotide CP-35 (SEQ ID NO: 153) was used with AP (SEQID NO: 21) on the double-stranded tinkered cDNA in a primary PCRreaction carried out as described herein. CP-35 has the sequence5′-GCTTCGGTACAATCATGTTT-3 (SEQ ID NO: 153), wherein T at position 3 canalso be C; G at position 6 can also be A, T or C; A at position 9 canalso be G; A at position 12 can also be G; A at position 15 can be G;and T at position 18 can also be C; this degenerate oligonucleotidesequence is the non-coding strand sequence corresponding to codingstrand sequence substantially encoding amino acids LysHisAspCysThrGluAla(SEQ ID NO: 154) of Cry j II (amino acids 71 to 77 of SEQ ID NO: 134;FIG. 28). Five percent (5 μl) of this initial amplification, designatedJC136, was then used in a secondary amplification with 100 pmoles eachof AP (SEQ ID NO: 21) and degenerate Cry j II primer CP-36, aninternally nested Cry j II oligonucleotide primer with the sequence5′-GGCTGCAGGTACAATCATGTTTGCCATC-3′ (SEQ ID NO: 155) wherein A atposition 11 can also be G; A at position 14 can also be G; A at position17 can also be G; T at position 20 can also be C; G at position 23 canalso be A, T, or C; and A at position 26 can also be G. The nucleotides5′-GGCTGCAG-3′ (SEQ ID NO: 156) (bases 1 through 8 of CP-36 (SEQ ID NO:155)) represent a Psi I restriction site added for cloning purposes. Theremaining degenerate oligonucleotide sequence of CP-36 (SEQ ID NO: 155)is the non-coding strand sequence corresponding to coding strandsequence substantially encoding amino acids AspGlyLysHisAspCysThr (SEQID NO: 157) of Cry j II (amino acids 69 to 75 of (SEQ ID NO: 134; FIG.28). The dominant amplified product, designated JC137, was a DNA band ofapproximately 265 base pairs, as visualized on an EtBr-stained 2% GTGagarose gel.

Amplified DNA was recovered by sequential chloroform, phenol, andchloroform extractions, followed by precipitation at −20° C. with 0.5volumes of 7.5 ammonium acetate and 1.5 volumes of isopropanol. Afterprecipitation and washing with 70% ethanol, the DNA was simultaneouslydigested with Xba I and Psi I in a 15 μl reaction and electrophoresedthrough a preparative 2% GTG SeaPlaque low melt gel (FMC, Rockport,Me.). The appropriate sized DNA band was visualized by EtBr staining,excised, and ligated into appropriately digested pUC19 for sequencing bythe dideoxy chain termination method (Sanger et al. (1977) Proc. NatlAcad Sci. USA 74: 5463-5476) using a commercially available sequencingkit (Sequenase kit, U.S. Biochemicals, Cleveland, Ohio).

The clones designated pUC19JC137a, pUC19JC137b, and pUC19JC137e werefound to contain sequences encoding the amino terminus of Cry j II. Allthree clones had identical sequence in their regions of overlap,although all three clones had different lengths in the 5′ untranslatedregion. Clone pUC19JC137b was the longest clone. The translated sequenceof these clones had complete identity to the disclosed 10 amino acidsequence of Cry j II (Sakaguchi et al., supra.), as well as to the Cry jII amino acid sequence described in Example 14. Amino acid numbering isbased on the sequence of the full length protein; amino acid 1corresponds to the initiating methionine (Met) of Cry j II. The positionof the initiating Met was supported by the presence of an upstreamin-frame-stop codon and by 78% homology of the surrounding nucleotidesequence with the plant consensus sequence that encompasses theinitiating Met, as reported by Lutcke et al. (1987) EMBO J. 6:43-48.

The cDNA encoding the remainder of Cry j II gene was cloned from thelinkered cDNA by using oligonucleotides CP-37 (which has the sequence5′-ATGTTGGACAGTGTTGTCGAA-3′ (SEQ ID NO: 158)) and AP (SEQ ID NO: 21) ina primary PCR, designated JC138ii. Oligonucleotide CP-37 (SEQ ID NO:158) corresponds to nucleotides 129 to 149 of SEQ ID NO: 133; FIG. 28,and is based on the nucleotide sequence determined for the partial Cry jII clone pUC19JC137b.

A secondary PCR reaction was performed on 5% of the initialamplification mixture, with 100 pmoles each of AP (SEQ ID NO: 21) andCP-38 (which has the sequence 5′-GGGAATTCAGAAAAGTTGAGCATTCTCGT-3′ (SEQID NO: 159)), the nested primer. The nucleotide sequence 5′-GGGAATTC-3′(SEQ ID NO: 159) (bases 1 through 8 of CP-38 (SEQ ID NO: 162))represents an Eco RI restriction site added for cloning purposes. Theremaining oligonucleotide sequence corresponds to nucleotides 177 to 197of SEQ ID NO: 133; FIG. 28, and is based on the nucleotide sequencedetermined for the partial Cry j II clone pUC19JC137b. The amplified DNAproduct, designated JC140iii, was purified and precipitated as above,followed by digestion with Eco RI and Asp 718 and electrophoresisthrough a preparative 1% low melt gel. The dominant DNA band, which wasapproximately 1.55 kb in length, was excised and ligated into pUC19 forsequencing. DNA was sequenced by the dideoxy chain termination method(Sanger et al. supra) using a commercially available kit (sequenase kit(U.S. Biochemicals, Cleveland, Ohio). Both strands were completelysequenced using M13 forward and reverse primers (N.E. Biolabs, Beverly,Mass.) and internal sequencing primers CP-35 (SEQ ID NO: 153), CP-38(SEQ ID NO: 159), CP-40 (SEQ ID NO: 161), CP-41 (SEQ ID NO: 162), CP-42(SEQ ID NO: 163), CP-43 (SEQ ID NO: 164), CP-44 (SEQ ID NO: 165), CP-45(SEQ ID NO: 166), CP-46 (SEQ ID NO: 167), CP-47 (SEQ ID NO: 168), CP-48(SEQ ID NO: 169), CP-49 (SEQ ID NO: 170), CP-50 (SEQ ID NO: 171), andCP-51 (SEQ ID NO: 172). CP-40 has the sequence 5′-GTTCTTCAATGGGCCATGT-3′(SEQ ID NO: 161) and corresponds to nucleotides 359 to 377 of SEQ ID NO:133; FIG. 28. CP-41 has the sequence 5′- GTGTTRAGGACT-GTCTCTCGG-3′ (SEQID NO: 162), which is the non-coding strand sequence that corresponds tonucleotides 720 to 739 of SEQ ID NO: 133; FIG. 28. CP-42 has thesequence 5′-TGTCCAGGCCATGGAATAAG-3′ (SEQ ID NO: 163), which correspondsto nucleotides 864 to 883 of SEQ ID NO: 133; FIG. 28 except that thefirst nucleotide was synthesized as a T rather than the correct G. CP43has the sequence 5′-GCCTTACATGGACTGCAACC-3′ (SEQ ID NO: 164), which isthe non-coding strand sequence that corresponds to nucleotides 1476 to1495 of SEQ ID NO: 135; FIG. 28. CP-44 has the sequence5′-TCCACGGGTCTGATAATCCA-3′, (SEQ ID NO: 165) which corresponds tonucleotides 612 to 631 of SEQ ID NO: 133; FIG. 28. CP-45 has thesequence 5′-AGGCAGGAAGCAATAACCC-3′ (SEQ ID NO: 166), which is thenon-coding strand sequence that corresponds to nucleotides 1254 to 1273of SEQ ID NO: 133; FIG. 28. CP-46 has the sequence5′-TACTGCACTTCAGCT-TCTGC-3′ (SEQ ID NO: 167), which corresponds tonucleotides 1077 to 1096 of SEQ ID NO: 133; FIG. 28. CP-47 has thesequence 5′-GGGGGTCTCCGAATTTATCA-3′, (SEQ ID NO: 168) which is thenon-coding strand sequence that substantially corresponds to nucleotides1039 to 1058 of SEQ ID NO: 133; FIG. 28, except that the fifthnucleotide of CP-47 was synthesized as a G rather than the correctnucleotide, T. CP48, which has the sequence 5′-GGATATTTCAGTGGACACGT-3′(SEQ ID NO: 169), corresponds to nucleotides 1290 to 1309 of SEQ ID NO:133; FIG. 28. CP-49 has the sequence 5′-TATTAGAAGACC-CTGTGCCT-3′ (SEQ IDNO: 170), which is the non-coding strand sequence that corresponds tonucleotides 821 to 840 of SEQ ID NO: 133; FIG. 28. CP-50 has thesequence 5′-CCATGTAAGGCCAAGTTAGT-3′ (SEQ ID NO: 171), which correspondsto nucleotides 1485 to 1504 of SEQ ID NO: 133; FIG. 28. CP-51 has thesequence 5′-ACACCATTACCCATTAGAGT-3′, (SEQ ID NO: 172) which is thenon-coding strand sequence that corresponds to nucleotides 486 to 505 ofSEQ ID NO: 133; FIG. 28.

Three clones, designated pUC19JC140iiia, pUC19JC140iiid andpUC19JC140iiie, were subsequently found to contain partial Cry j IIsequence. The sequence of clone pUC19JC140iiid was chosen as theconsensus sequence since it had the longest 3′ untranslated region. Thesequences of pUC19JC140iiid and pUC19JC137b were used to construct thecomposite Cry j II sequence shown in FIG. 28 (SEQ ID NO: 133). In thiscomposite, nucleotide 230 is reported as the A found in pUC19JC137b(also, pUC19JC137a, pUC19JC140iiia and pUC19JC140iiie) not as the Gfound in pUC19JC140iiid; however both A and G at nucleotide 230 encodeLys at amino acid 63 (SEQ ID NO: 134). The sequence of clonepUC19JC140iiia was identical to that of pUC19JC140iiid except for thefollowing: pUC19JC140iiia has a T at nucleotide 357 in place of a C (nopredicted change in amino acid 106), has C at nucleotide 754 instead ofT (changes amino acid 238 from Ile to Thr), C at nucleotide 1246 insteadof T (changes amino acid 402 from Leu to Pro), and T at nucleotide 1672instead of C (untranslated region). The sequence of clone pUC19JC140iiiewas identical to that of pUC19JC140iiid except for G at nucleotide 794instead of A (changes amino acid 251 from Ile to Met), and T atnucleotide 357 in place of C (no predicted change in amino acid 106).

An earlier attempt at cloning the JC140iii PCR product using an EcoRI/Xba I digest (oligonucleotide AP has both Xba I and Asp 718restriction enzyme sites) yielded cDNA that was cut in half due to aninternal Xba I restriction site in the Cry j II cDNA, giving rise to 800and 750 bp bands; the 750 bp band was succesfully cloned into Eco RI/XbaI digested pUC19 and sequenced. Two 750 bp clones were sequenced andfound to be the 5′ half of the Cry j II molecule: clones pUC19JC140-2aand pUC19JC140-2b. Clone pUC19JC140-2a has C for nucloeotide 297 insteadof T (changes amino acid 86 from Cys to Arg) and clone pUC19JC140-2b hasG for nucleotide 753 instead of A (changes amino acid 238 from Ile toVal). Both clone pUC19JC140-2a and clone pUC19JC140-2b have a T atnucleotide 357 in place of C (no predicted change in amino acid 106).

Two different PCR amplifications were also sequenced directly to verifythe clonal Cry j II sequence using the Amplitaq Cycle Sequencing kit(Perkin Elmer Cetus, Norwalk, Conn.). This procedure involves the[³²P]-end-labelling of oligonucleotide sequencing primers which are thenannealled (1.6 pmoles in 1 μl) to template DNA and elongated withdideoxy NTPs (methodology of Sanger et al. (1977) Proc. Natl. Acad Sci.USA 74:5463-5476) in a PCR reaction also containing 4 μl 10× Cycling Mix(contains 0.5 U/μl Amplitaq DNA Polymerase), 5 μl template DNA (10-100fmoles) and dH₂O to 20 μl. The dGTP in the termination mixes in this kithave been replaced by 7-deaza-dGTP, which provides increased resolutionof sequences containing high G+C regions of DNA. The template DNA was aPCR product that was recovered by sequential chloroform, phenol, andchloroform extractions, precipitated at −20° C. with 0.5 volumes of 7.5ammonium acetate and 1.5 volumes of isopropanol, then electrophoresedthrough a preparative 1 or 2% SeaPlaque low melt gel (FMC). Appropriatesized DNA bands were visualized by EtBr staining, excised, and treatedwith Gelase (Epicentre Technologies, Madison, Wis.) to remove theagarose. The DNA was again precipitated, and resuspended in 50 μl TE (10mM Tris, pH 7.4, 1 mM EDTA, pH 8.0) containing 20 μg/1 ml RNAse(Boehringer Mannheim, Indianapolis, Ind.). Two secondary amplificationswhich had been used to clone Cry j II were repeated, and used astemplate DNA for PCR cycle sequencing: JC137ii, the 5′ end PCR,(amplified from the 1° PCR JC136 above) was reamplified Witholigonucleotides AP and CP-36; and JC140ii, the 3′ end PCR, (amplifiedfrom the 1° PCR JC138ii above) was reamplified with oligonucleotides APand CP-38. Both of the 1° amplifications used were precipitated,electrophoresed through a preparative 1 or 2% SeaPlaque low melt gel(FMC), and the appropriate sized bands were visualized by EtBr stainingand excised. Two μl of each 1° amplification was then used in thecorresponding 2° PCR reaction. The 2° PCR product was then prepared asDNA template for PCR cycle sequencing as described above. Theoligonucleotides used as primers in PCR cycle sequencing, many of whichwere used to sequence the clones, are as follows: for JC137ii, CP-36(SEQ ID NO: 155) and CP-39, which has the sequence5′-CTGTCCAACATAATTTGGGC-3′ (SEQ ID NO: 173) and is the non-coding strandsequence corresponding to nucleotides 120 to 139 of SEQ ID NO: 133; FIG.28. The oligonucleotide primers used for sequencing JC140ii were CP-38(SEQ ID NO: 159), CP-40 (SEQ ID NO: 161), CP-41 (SEQ ID NO: 162), CP-42(SEQ ID NO: 163), CP-43 (SEQ ID NO: 164), CP-44 (SEQ ID NO: 165), CP-45(SEQ ID NO: 166), CP-46 (SEQ ID NO: 167), CP-47 (SEQ ID NO: 168), CP-49(SEQ ID NO: 170), CP-50 (SEQ ID NO: 171), CP-54 (SEQ ID NO: 173), whichhas the sequence 5′-CATGGCAGGGTGGTTCAGGC-3′ (SEQ ID NO: 173),corresponds to nucleotides 985 to 1004 of SEQ ID NO: 133; FIG. 28,CP-55, which has the sequence 5′-TAGCCCCATTTACGTGCACG-3′ (SEQ ID NO:174) and is the non-coding strand sequence that corresponds tonucleotides 929 to 948 of SEQ ID NO: 133; FIG. 28, and CP-56, which hasthe sequence 5′-TTGGGGTCGAGGCCTCCGAA-3′ (SEQ ID NO: 175) and correspondsto nucleotides 1437 to 1456 of SEQ ID NO: 133; FIG. 28. The sequence ofthis full-length PCR cycle sequencing had only 2 nucleotide changes fromthe composite pUC19JC137b/pUC19JC140iiid Cry j II sequence shown in FIG.28 (SEQ ID NO: 133), neither of which lead to an amino acid change.There was a T instead of C at nucleotide 357 (no predicted change inamino acid 106), and a C instead of A at nucleotide 635 (no amino acidchange).

The nucleotide and predicted amino acid sequences of Cry j II are shownin FIGS. 28 and 29 (SEQ ID NO: 133 and 134). This is a compositenucleotide sequence from the two overlapping clones pUC19JC137b andpUC19JC140iiid. Sequencing of multiple independent clones and cyclesequencing of PCR product confirmed the nucleotide sequence of FIG. 4(SEQ ID NO: 133). There were several nucleotide changes resulting inpredicted amino acid changes, as cited above. However, all nucleotidepolymorphisms, with the exception of the T for C substitition atnucleotide 357, were only observed in single clones or sequencingreactions. Although T was seen at nucleotide 357 in all clones exceptpUC19JC140iiid, both C and T encode Leu at amino acid 106.

The complete cDNA sequence for Cry j II is composed of 1726 nucleotides,including 41 nucleotides of 5′ untranslated sequence, an open readingframe of 1542 nucleotides starting with the codon for an initiating Met(nucleotides 42-44 of SEQ ID NO: 133; FIG. 28), and a 143 bp 3′untranslated region. There is a consensus polyadenylation signalsequence in the 3′ untranslated region 64 nucleotides 5′ to the poly Atail (nucleotides 1654-1659 of SEQ ID NO: 133; FIG. 28). The position ofthe initiating Met is confirmed by the presence of an in-frame upstreamstop codon and by 78% homology with the plant consensus sequence thatencompasses the initiating Met (TAAAAUGGC (bases 38 through 46 of (SEQID NO: 133); FIG. 28) found in Cry j II compared with the AACAAUGGCconsensus sequence for plants, Lutcke et al. (1987) EMBO J. 6: 4348).The open reading frame encodes a deduced protein of 514 amino acids thathas complete sequence identity with the published partial proteinsequence for Cry j II (Sakaguchi et al. supra), which corresponds toamino acids 55 through 64 of SEQ ID NO: 134; FIG. 28. The predicted Cryj II protein has 20 Cys, contains four potential N-linked glycosylationsites corresponding to the consensus sequence N—X—S/T, has a predictedmolecular weight of 56.6 kDa and a predicted pI of 9.08.

Detection of three separate NH₂ termini sequences for Cry j II (the longform and the short form as determined in Example 14 and the NH₂ terminusdetermined by Sakaguchi et al., supra, as shown in FIG. 6) may suggestthat the amino terminus of the mature Cry j II protein is blocked andthat the sequences obtained by sequence analysis of purified proteinrepresent proteolytic cleavage products. As shown in FIG. 6, the aminoacid sequence of the long form of Cry j II begins at amino acid 46 andthe amino acid sequence of the short form of Cry j II begins at aminoacid 51; and the NH2-terminal sequence determined by Sakaguchi et al.begins at amino acid 54. It is also possible that amino acids 1 to 45represent the leader/pre-pro position of Cry j II that is enzymaticallycleaved to give a functionally active protein beginning at amino acid 46of SEQ ID NO: 134; FIG. 28. The sequences beginning at amino acids 51and 54 represent breakdown products of the protein beginning at aminoacid 46. There is a predicted cleavage site between amino acids 22 and23 of SEQ ID NO: 134; FIG. 28 using the method of von Heijne (NucleicAcids Res. (1986) 14:4683-4690). If the mature Cry j II protein startedat amino acid 23 of SEQ ID NO: 134; FIG. 28, the protein would be 492amino acids long with a predicted molecular weight of 54.2 kDa and apredicted pI of 9.0.

Searching the Swiss-Prot data base with the Cry j II sequencedemonstrated that Cry j II is 43.3% homologous (33.3% identical topolygalacturonase of tomato (Lycopersicon esculentum) and 48.4%homologous (32.6% identical) to polygalacturonase of corn, Zea mays. Allnucleotide and amino acid sequence analyses were performed using PCCENE(Intelligenetics, Mountain View, Calif.).

EXAMPLE 16

Extraction of RNA from Japanese Cedar Pollen Collected in Japan andExpression of Recombinant Cry j II

Fresh pollen collected from a pool of Cryptomeria japonica (Japanesecedar) trees in Japan was frozen immediately on dry ice. RNA wasprepared from 500 mg of the pollen, essentially as described by Frankisand Mascarenhas Ann. Bot. 45:595-599. The samples were ground by mortarand pestle on dry ice and suspended in 5 ml of 50 mM Tris pH 9.0 with0.2 M NaCl, 1 mM EDTA, 1% SDS that had been treated overnight with 0.1%DEPC. After five extractions with phenol/chloroform/isoamyl alcohol(mixed at 25:24:1), the RNA was precipitated from the aqueous phase with0.1 volume 3 M sodium acetate and 2 volumes ethanol. The pellets wererecovered by centrifugation, resuspended in 2 ml dH₂O and heated to 65°C. for 5 minutes. Two ml of 4 M lithium chloride were added to the RNApreparations and they were incubated overnight at 0° C. The RNA pelletswere recovered by centrifugation, resuspended in 1 ml dH₂O, and againprecipitated with 3 M sodium acetate and ethanol overnight. The finalpellets were resuspended in 100 μl dH₂O and stored at −80° C.

Double stranded cDNA was synthesized from 8 μg pollen RNA using the cDNASynthesis Systems kit (BRL) with oligo dT priming according to themethod of Gubler and Hoffman (1983) Gene 25:263-269. PCRs were carriedout using the GeneAmp DNA Amplification kit (Perkin Elmer Cetus) whereby10 μl 10× buffer containing dNTPs was mixed with 100 pmol each of asense oligonucleotide and an anti-sense oligonucleotide, cDNA (10 μl ofa 400 μl double stranded cDNA reaction mix), 0.5 μl Amplitaq DNApolymerase, and distilled water to 100 μl.

The samples were amplified with a programmable thermal controller fromMJ Research, Inc. (Cambridge, Mass.). The first 5 rounds ofamplification consisted of denaturation at 94° C. for 1 min, annealingof primers to the template at 45° C. for 1 min, and chain elongation at72° C. for 1 min. The final 20 rounds of amplification consisted ofdenaturation as above, annealing at 55° C. for 1 min, and elongation asabove.

A new set of primer pairs was synthesized for amplification of a Cry jII cDNA from the initiating Met to the stop codon. CP-52 has thesequence 5′-GCCGAATTCATGGCCATGAAATTAATT-3′ (SEQ ID NO: 179) where thenucleotide sequence 5′-GCCGAATTC-3′ (SEQ ID NO: 180) (bases I through 9of CP-52 (SEQ ID NO: 179) represents an Eco RI restriction site addedfor cloning purposes, and the remaining sequence corresponds tonucleotides 42 to 59 of SEQ ID NO: 133; FIG. 28. CP-53 has the sequence5′-CGGGGATCCTCATTATGGATG-GTAGAT-3′ (SEQ ID NO: 181) where the nucleotidesequence 5′-CGGGGATCC-3′ (SEQ ID NO: 182) (bases 1 through 9 of CP-53(SEQ ID NO: 181)) represents a Bam HI restriction site added for cloningpurposes, and the remaining oligonucleotide sequence of CP-53 (SEQ IDNO: 181) is complementary to coding strand sequence corresponding tonucleotides 1572 to 1589 of SEQ ID NO: 133; FIG. 28. The PCR reactionwith CP-52 (SEQ ID NO: 179) and CP-53 (SEQ ID NO: 181) on the doublestranded Japanese Cedar pollen cDNA yielded a band of approximately 1.55kb on an EtBr-stained agarose minigel, and was called JC145. AmplifiedDNA was recovered by sequential chloroform, phenol, and chloroformextractions, followed by precipitation at −20° C. with 0.5 volumes of7.5 ammonium acetate and 1.5 volumes of isopropanol. After precipitationand washing with 70% ethanol, the DNA was simultaneously digested withEco RI and Bam HI in a 15 μi reaction, and electrophoresed through apreparative 1% SeaPlaque low melt gel (FMC). Appropriate sized DNA bandswere visualized by EtBr staining, excised, and ligated intoappropriately digested pUC19 for sequencing by the dideoxy chaintermination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA74:5463-5476) using a commercially available sequencing kit (Sequenasekit, U.S. Biochemicals, Cleveland, Ohio).

Clones pUC19JC145a and pUC19JC145b were completely sequenced using M13forward and reverse primers (N.E. Biolabs, Beverly, Mass.) and internalsequencing primers CP-41 (SEQ ID NO: 162), CP-42 (SEQ ID NO: 163), CP-44(SEQ ID NO: 165), CP-46 (SEQ ID NO: 167), and CP-51 (SEQ ID NO: 172).The nucleotide and deduced amino acid sequences of clones pUC19JC145aand pUC19JC145b were identical to the Cry j II sequence of FIG. 28 (SEQID NO: 133 and 134), with the following exceptions. Clone pUC19JC145awas found to contain a single nucleotide difference from the previouslyknown Cry j II sequence: it has a C at nucleotide position 1234 of SEQID NO: 133; FIG. 28 rather than the previously described T. Thisnucleotide change results in a predicted amino acid change from Ile toThr at amino acid 398 of the Cry j II protein (SEQ ID NO: 134). ClonepUC19JC145b has a G at nucleotide position 1088 of SEQ ID NO: 133; FIG.28 rather than the previously described A, and an A for a G atnucleotide 1339. The nucleotide change at 1088 is silent and does notresult in a predicted amino acid change. The nucleotide change atposition 1339 results in a predicted amino acid change from Ser to Asnat amino acid 433 of the Cry j II protein. None of these polymorphismshave yet been confirmed by independently-derived PCR clones or by directamino acid sequencing and may be due to the inherent error rate of Taqpolymerase (approximately 2×10⁻⁴, Saiki et al. (1988) Science239:487-491). However, such polymorphisms in primary nucleotide andamino acid sequences are expected.

Expression of Cry j II was performed as follows. Ten μg of pUC19JC145bwas digested simultaneously with Eco RI and Bam HI. The nucleotideinsert encoding Cry j II (extending from nucleotide 42 through 1589 of(SEQ ID NO: 133) FIG. 28) was isolated by electrophoresis of this digestthrough a 1% SeaPlaque low melt agarose gel. The insert was then ligatedinto the appropriately digested expression vector pET-11d (Novagen,Madison, Wis.; Jameel et al. (1990) J. Virol. 64:3963-3966) modified tocontain a sequence encoding 6 histidines (His 6) immediately 3′ of theATG initiation codon followed by a unique Eco RI endonucleaserestriction site. A second Eco RI endonuclease restriction site in thevector, along with neighboring Cla I and Hind III endonucleaserestriction sites, had previously been removed by digestion with Eco RIand Hind III, blunting and religation. The histidine (His₆) sequence wasadded for affinity purification of the recombinant protein (Cry j I) ona Ni²⁺ chelating column (Hochuli et al. (1987) J. Chromatog.411:177-184; Hochuli et al. (1988) Bio/Tech. 6:1321-1325.). Arecombinant clone was used to transform Escherichia coli strainBL21-DE3, which harbors a plasmid that has anisopropyl-β-D-thiogalactopyranoside (IPTG)-inducible promoter precedingthe gene encoding T7 polymerase. Induction with IPTG leads to highlevels of T7 polymerase expression, which is necessary for expression ofthe recombinant protein in pET-11d. Clone pET-11dΔHRhis₆JC145b.a wasconfirmed to be a Cry j II clone in the correct reading frame forexpression by dideoxy sequencing (Sanger et al. supra) with CP-39.

Expression of the recombinant protein was examined in an initial smallculture. An overnight culture of clone pET-11dΔHRhis₆JC145b.a was usedto innoculate 50 ml of media (Brain Heart Infusion Media, Difco)containing ampicillin (200 μg/ml), grown to an A₆₀₀=1.0 and then inducedwith IPTG (1 mM, final concentration) for 2 hrs. One ml aliquots of thebacteria were collected before and after induction, pelleted bycentrifugation, and crude cell lysates prepared by boiling the pelletsfor 5 minutes in 50 mM Tris HCl, pH 6.8, 2 mM EDTA, 1% SDS, 1%β-mercaptoethanol, 10% glycerol, 0.25% bromophenol blue (Studier et al.,(1990) Methods in Enzymology 185:60-89). Recombinant protein expressionwas examined on a 12% Coomassie blue-stained SDS-PAGE gel, according tothe method in Sambrook et al., supra, on which 25 μl of the crudelysates were loaded. A negative control consisted of crude lysate fromuninduced bacteria containing the plasmid with Cry j II. There was nonotable increase in production of any recombinant E. coli protein in therange of 58 Kd, the size predicted for the recombinant Cry j II with theHis₆ leader.

The pET-11dΔHRhis₆JC145b.a clone was then grown on a larger scale toexamine if there was any recombinant protein being expressed. A 2 mlculture of bacteria containing the recombinant plasmid was grown for 8hr, then 3 μl was spread onto each of 6 (100×15 mm) petri plates with1.5% agarose in LB medium (Gibco-BRL, Gaithersburg, Md.) containing 200μg/ml ampicillin, grown to confluence overnight, then scraped into 6 Lof liquid media (Brain Heart Infusion media, Difco) containingampicillin (200 μg/ml). The culture was grown until the absorbance atA600 was 1.0, IPTG added (1 mM final concentration), and the culturegrown for an additional 2 hours.

Bacteria were recovered by centrifugation (7,930×g, 10 min) and lysed in50 ml of 6M Guanidine-HCl, 0.1M Na₂HPO₄, pH 8.0, for 1 hour withvigorous shaking. Insoluble material was removed by centrifugation(11,000×g, 10 min, 4° C.). The pH of the lysate was adjusted to pH 8.0,and the lysate applied to a 50 ml Nickel NTA agarose column (Qiagen)that had been equilibrated with 6 M Guanidine HCl, 100 mM Na₂HPO₄, pH8.0. The column was sequentially washed with 6 M Guanidine HCl, 100 mMNa₂HPO₄, 10 mM Tris-HCl, pH 8.0, then 8 M urea, 100 mM Na₂HPO₄, pH 8.0,and finally 8 M urea, 100 mM sodium acetate, 10 mM Tris-HCl, pH 6.3. Thecolumn was washed with each buffer until the flow through had anA₂₈₀≦0.05.

The recombinant Cry j II protein was eluted with 8 M urea, 100 MM sodiumacetate, 10 mM Tris-HCl, pH 4.5, and collected in 10 ml aliquots. Theprotein concentration of each fraction was determined by A₂₈₀ and thepeak fractions pooled. An aliquot of the collected recombinant proteinwas analyzed on SDS-PAGE according to the method in Sambrook et al.supra.

This 6L prep, JC11pET-1, yielded 1.5 mg of recombinant Cry j II, whichwas resolved into 2 major bands on SDS-PAGE at 58 kDa and 24 kDa. The 58kDa band, which represents recombinant Cry j II, was approximately 9-10%of the total protein as determined by densitometry measurement (ShimadzuFlying Spot Scanner, Shimadzu Scientific Instruments, Inc., Braintree,Mass.). The 24 kDa band accounts for about 90% of the total protein andmay represent a degradation product of the recombinant Cry j II or an E.coli contaminant.

Another Cry j II expression construct was made by the ligation of thepUC19JC140iiid Cry j II insert into appropriately digested pET11dΔHR(with the 6 histidine leader). The vector was derived from anotherpET11dΔHR construct whose insert supplied an EcoR I site (at the 5′pET11dΔHR-insert junction) and an Asp 718site (at the 3′ end of theinsert); the construct was digested with these two enzymes, run on a lowmelt minigel as above, and the vector recovered as a band in low meltagarose. The pUC19JC140iiid construct was digested with Eco R I and Asp718 to release the Cry j II insert, which was isolated on a low meltminigel and ligated into the Eco R UAsp 718 digested pET11dΔHR vectorprepared above. Five clones were found to contain the correct nucleotidesequence at the insert/vector 5′ junction, when sequenced by dideoxysequencing (as above) with CP-39. This new construct, when expressed,would begin at amino acid 46 of Cry j II as shown in FIGS. 28 and 29.This recombinant protein is designated rCry j II Δ46. A 50 ml smallscale expression test (as performed above) showed that the expressionlevel of rCry j II Δ46 from this construct, designatedpET11dΔHRJC140iiid2, would be much greater than the initial expressionlevel from pET11dΔHRJC145b2. A 9L prep, JC11pET-3, was processed asabove, and yielded 200 mg of rCry j II Δ46 at 80% purity as determinedby densitometry of a Coomasie blue stained 12% SDS-PAGE gel.

EXAMPLE 17

Northern Blot on RNA from Japanese Cedar Pollen Sources

A northern blot analysis was performed on the RNA isolated from JapaneseCedar pollen from both the Arnold Arboretum tree and the pooled treesfrom Japan. Using essentially the method of Sambrook, supra, ten μg ofRNA isolated from Japanese cedar pollen collected from the ArnoldArboretum (Boston, Mass.) and 15 μg pooled RNA from Japanese cedarpollen collected from trees in Japan were run on a 1.2% agarose gelcontaining 38% formaldehyde and 1×MOPS (20×=0.4M MOPS, 0.02M EDTA, 0.1MNaOAc, pH 7.0) solution. The RNA samples (first precipitated with 1/10volume sodium acetate, 2 volumes ethanol to reduce volume andresuspended in 5.5 μl dH2O) were run with 10 μl formaldehyde/formamidebuffer containing loading dyes with 15.5% formaldehyde, 42% formamide,and 1.3×MOPS solution, final concentration. The samples were transferredto Genescreen Plus (NEN Research Products, Boston, Mass.) by capillarytransfer in 10×SSC (20×=3M NaCl, 0.3M Sodium Citrate), after which themembrane was baked 2 hrs at 80° C. and UV irradiated for 3 minutes.Prehybridization of the membrane was at 60° C. for 1 hour in 4 ml 0.5MNaPo4 (pH 7.2), 1 mM EDTA, 1% BSA, and 7% SDS. The antisense probe wassynthesized by asymmetric PCR on the JC 145 amplification in low meltagarose (above), where 2 μl DNA is amplified with 2 μl dNTP mix (0.167mM dATP, 0.167 mM dTTP, 0.167 mM dGTP, and 0.033 mM dCTP), 2 μl 10×PCRbuffer, 10 μl ³²P-dCTP (100 μCi; Amersham, Arlington Heights, Ill.), 1μl (100 pmoles) antisense primer CP-53, 0.5 μl Taq polymerase, and dH2Oto 20 μl; the 10×PCR buffer, dNTPs and Taq polymerase were from PerkinElmer Cetus (Norwalk, Conn.). Amplification consisted of 30 rounds ofdenaturation at 94° C. for 45 sec, annealing of primer to the templateat 60° C. for 45 sec. and chain elongation at 72° C. for 1 min. Thereaction was stopped by addition of 100 μl TE, and the probe recoveredover a 3 cc G-50 spin column (2 ml G-50 Sephadex [Pharmacia, Uppsala,Sweden] in a 3 cc syringe plugged with glass wool, equilibrated with TE)and counted on a 1500 TriCarb Liquid Scintillation Counter (Packard,Downers Grove, Ill.). The probe was added to the prehybridizing bufferat 10⁶ cpm/ml and hybridization was carried out at 60° C. for 16 hrs.The blot was washed in high stringency conditions: 3×15 min at 65° C.with 0.2%SSC/1% SDS, followed by wrapping in plastic wrap and exposureto film at −80° C. A seven hour exposure of this Northern blot analysisrevealed a single thick band at approximately 1.7 kb for both RNAcollected from the Arboretum tree and the RNA collected from the pooledtrees from Japan. This message is the expected size for Cry j II aspredicted by PCR analysis of the cDNA.

EXAMPLE 18

Direct Binding Assay of IgE to Cry j I, Cry j H and Recombinant Cry jII.

Costar assay plates were coated with Cry j I or Cry j II at 2 μg/mL orrecombinant Cry j II preparation at 10 μg/mL (approximately 20% pure) ina volume of 50 μL overnight at 4° C. The coating antigens were removedand the wells were blocked with 0.5% gelatin, PVP (polyvinyl pyrolidine)1 mg/mL in PBS, 200 μL/well for 2 hours at room temperature. Theanti-Cry j I monoclonal antibody, 4B11, was serially diluted inPBS-Tween 20 starting at a 1:1000 dilution. The human plasma wereserially diluted in PBS-Tween at a starting dilution of 1:2. For thisset 23 plasma samples from patients symptomatic for Japanese cedarpollen allergy chosen for IgE binding analysis. The first antibodyincubation proceeded overnight at 4° C. Following three washes withPBS-Tween the second antibodies were added (biotinylated goat anti-mouseIg or goat anti-human IgE both at 1:2000) and incubated for two hours atroom temperature at 100 μL/well. After washing 3 times, 100 μL of TMBsubstrate was added per well (Kirkgaard Perry Labs). This solution wasremoved and streptavidin-HRPO diluted to 1:10,000, was added at 100μL/well. The color was allowed to develop for 2-5 minutes. The reactionwas stopped by the addition of 100 μL/well of 1M phosphoric acid. Plateswere read on a Microplate IL310 Autoreader (Biotek Instruments,Winooski, Vt.) with a 450 nm filter. The absorbance levels of duplicatewells were averaged. The graphed results (log of the dilution vs.absorbance) of the ELISA assays are shown in FIGS. 31 to 39. The summaryof the results are given in FIG. 40. A positive binding result,indicated by a plus sign is determined to be a reading of two-fold orgreater above background (no first antibody) at the second dilution ofplasma (1:6).

In FIG. 31 the binding response of the monoclonal antibody, 4B11, andseven patients' (Batch 1) plasma IgE is shown to purified Cry j I as thecoating antigen. The monoclonal antibody, raised against purified Cry jI shows a saturating level of binding for the whole dilution series. Theindividual patient samples show a variable response of IgE binding tothe Cry j I preparation. One patient, #1034, has no detectable bindingto this protein preparation. All the patient samples were obtained fromindividuals claiming to be symptomatic for Japanese cedar pollen allergyand the results of their MAST scores are shown in FIG. 40. FIG. 32 is agraph representing the binding of the same antibody set as in FIG. 31 topurified native Cry j II. The anti-Cry j I monoclonal antibody, 4B11, isnegative on this preparation demonstrating lack of cross-reactivitybetween the two allergen antigens. In general, there is a lower overallresponse to this allergenic component of cedar pollen with more patientsamples showing decreased binding. However, patient #1034, that wasnegative on Cry j I shows very strong reactivity to Cry j II. In thelast antigen set, FIG. 33, using recombinant Cry j II (rCry j II),monoclonal antibody 4B11 reactivity is negative and there is furtherreduction in binding of the human IgE samples compared to biochemicallypurified Cry j II. Two of the patients, #1143 and #1146, are clearlypositive for IgE binding to the recombinant form of Cry j II althoughthe patient that reacted the strongest to biochemically purified form isnegative here, 1034. FIGS. 34-39 represent the application of the sameantigen sets for the direct binding analysis of the next sixteenpatients designated patient Batch 2 and patient Batch 3 in FIGS. 34-39.

The table shown in FIG. 40 summarizes both the MAST scores, performed inJapan on the plasma samples before shipment using a commerciallyavailable kit, and the direct ELISA results outlined above. Two patientswere negative by the MAST assay, however, one of these patients, #1143,was positive on all the ELISA antigens. The number of positive responsesfor each antigen is shown and this represents a measure relativeallergenicity of the different allergen preparations. These resultsdemonstrate that Cry j II is an allergen as defined by human allergicpatient IgE reactivity and that there are some patients who are notreactive to Cry j I but are reactive to Cry j II. The frequency ofresponse in this population of patients is less to Cry j II than to Cryj I.

EXAMPLE 19

Japanese Cedar Pollen Allergic Patient T Cell Studies with Cry j II andCry j II Peptides.

Synthesis of Cry j II Peptides

Japanese cedar pollen Cry j II peptides designated Cry j IIA (SEQ ID NO:185), Cry j IIB (SEQ ID NO: 186), Cry j IIG (SEQ ID NO: 191), Cry j IIH(SEQ ID NO: 192) and Cry j IIQ (SEQ ID NO: 193) were synthesized usingstandard Fmoc/tBoc synthetic chemistry and purified by Reverse PhaseHPLC. The amino acid sequence of peptide Cry j IIA is FTFKVDGIIAAYQ (SEQID NO: 185) which corresponds to amino acids 116-128 of SEQ ID NO: 134;FIGS. 28 and 41. The amino acid sequences of peptide Cry j IIB isNGYFSGHVIPACKN (SEQ ID NO: 186) which corresponds to amino acids 416-429of SEQ ID NO: 134; FIGS. 28 and 41. The amino acid sequence of Cry j IIGis shown in FIG. 41 and corresponds to amino acids 152-175 of SEQ IS NO:134, FIG. 28. The amino acid sequence of Cry j IIH is shown in FIG. 41and corresponds to amino acids 386-409 of SEQ ID NO: 134, FIG. 28. Theamino acid sequence of Cry j IIQ is shown in FIG. 41, and corresponds toamino acids 269-292 of SEQ ID NO: 134, FIG. 28. The amino acid sequencesof the peptide names are consistent throughout.

Japanese cedar pollen Cry j II peptides designated Cry j IIC (SEQ ID NO:187), Cry j IID (SEQ ID NO: 188), Cry j IIE (SEQ ID NO: 189), and Cry jIIF (SEQ ID NO: 190) having amino acid sequences as shown in FIG. 41were synthesized using recombinant techniques and expressed as discussedin Example 20. These peptides are modified peptides derived from thefull length amino acid sequence Cry j II (SEQ ID NO: 134) shown in FIG.28. Peptide Cry j IIC (SEQ ID NO: 187) corresponds to amino acids 46-163of SEQ ID NO: 134 shown in FIG. 28; peptide Cry j IID (SEQ ID NO: 188)corresponds to amino acids 164-280 of SEQ ID NO: 134 shown in FIG. 28;peptide Cry j IIE (SEQ ID NO: 189) corresponds to amino acids 281-396 ofSEQ ID NO: 134 shown in FIG. 28; and peptide Cry j IIF (SEQ ID NO: 190)corresponds to amino acids 397-514 of SEQ ID NO: 134 shown in FIG. 28.

T Cell Responses to Japanese Cedar Pollen Antigen Peptides

Peripheral blood mononuclear cells (PBMC) were purified by lymphocyteseparation medium (LSM) centrifugation of 60 ml of heparinized bloodfrom up to nine Japanese cedar pollen-allergic patients who exhibitedclinical symptoms of seasonal rhinitis and was MAST and/or skin testpositive for Japanese cedar pollen. Long term T cell lines wereestablished by stimulation of 2×10⁶ PBL/ml in bulk cultures of completemedium (RPMI-1640, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin,5×10⁻⁵M 2-mercaptoethanol, and 10 mM HEPES supplemented with 5% heatinactivated human AB serum) with 10 μg/ml of partially purified nativeCry j II for 7 days at 37° C. in a humidified 5% CO₂ incubator to selectfor Cry j II reactive T cells. This amount of priming antigen wasdetermined to be optimal for the activation of T cells from mostJapanese cedar pollen Cry j II allergic patients. Viable cells werepurified by LSM centrifugation and cultured in complete mediumsupplemented with 5 units recombinant human IL-2/ml and 5 unitsrecombinant human IL-4/ml for up to three weeks until the cells nolonger responded to lymphokines and were considered “rested”. Theability of the T cells to proliferate to peptides Cry j IIA (SEQ ID NO:185) and Cry j IIB (SEQ ID NO: 186), recombinant Cry j II (rCry j II)(SEQ ID NO: 134), purified native Cry j II, was then assessed. Forassay, 2×10⁴ rested cells were restimulated in the presence of 2×10⁴autologous Epstein-Barr virus (EBV)-transformed B cells (prepared asdescribed in Example 6) (gamma-irradiated with 25,000 RADS) with 2-50’g/ml of rCry j II (SEQ ID NO: 134), purified native Cry j II, peptidesCry j IIA (SEQ ID NO: 185) and Cry j IIB (SEQ ID NO: 186), positivecontrol (PHA), negative control (Amb a I.1), in a volume of 200 mlcomplete medium in duplicate or triplicate wells in 96-well round bottomplates for 2-4 days. The optimal incubation was found to be 3 days. Eachwell then received 1 μCi tritiated thymidine for 16-20 hours. The countsincorporated were collected onto glass fiber filter mats and processedfor liquid scintillation counting. The maximum response in a titrationof each peptide is expressed as the stimulation index (S.I.). The S.I.is the counts per minute (CPM) incorporated by cells in response topeptide, divided by the CPM incorporated by cells in medium only. Apositivity index may be calculated by multiplying the mean S.I.(indicated above each bar in FIGS. 42 and 43) by the percentage ofindividuals responding to the peptide (indicated in parentheses aboveeach bar in FIGS. 42 and 43). The results shown in FIG. 42 demonstratethat the Japanese cedar pollen allergic patients tested (n=6) respondwell to recombinant Cry j II, and purified native Cry j II, as expected.There was minimal cross reaction with negative control Amb a 1.1 wholeprotein as expected. The response to peptides Cry j IIA (SEQ ID NO: 185)and Cry j IIB (SEQ ID NO: 186) in a population of only six patients,indicates that it may be likely that epitopes exit within thesepeptides. Additional Japanese cedar pollen allergic patients will betested in this assay system and it is believed that these studies willshow that peptides Cry j IIA (SEQ ID NO: 185) and Cry j IIB (SEQ ID NO:186) contain T cell epitopes.

FIG. 43 shows T cell proliferative assays performed substantially asdescribed above with Cry j II reactive T cells from a total of 9Japanese Cedar pollen allergic patients. As shown in FIG. 43, these Tcell lines react not only to rCry j II, and purified native Cry j II asexpected, but also to peptides Cry j IIC (SEQ ID NO: 187), Cry j IID(SEQ ID NO: 188), Cry j IIE (SEQ ID NO: 189), and Cry j IIF (SEQ ID NO:190), Cry j IIG (SEQ ID NO: 191) and Cry j IIH (SEQ ID NO: 192). Therewas minimal cross reactivity with the negative control Amb a I.1 wholeprotein, as expected. The positive mean S.I. (indicated above each barin parentheses) for each peptide tested indicates that each peptidecontains at least one T cell epitope. Peptide fragments derived fromeach of peptides Cry j IIC (SEQ ID NO: 187), Cry j IID (SEQ ID NO: 188),Cry j IIE (SEQ ID NO: 189), and Cry j IIF (SEQ ID NO: 190) may besynthesized and used in the above-described T cell proliferation assaysystem to further analyze the location of each T cell epitope.

EXAMPLE 20

Recombinant Production of Peptide Subconstructs Designated Cry j IIC(SEQ ID NO: 187), Cry j IID (SEQ ID NO: 188), Cry j IIE (SEQ ID NO:189), and Cry j IIF (SEQ ID NO: 190)

Four Cry j II peptide subconstructs designated construct #1(Cry j IIC(SEQ ID NO: 187)), construct #2 (Cry j IID (SEQ ID NO: 188)), construct#3 (Cry j IIE (SEQ ID NO: 189)), and construct #4 (Cry j IIF (SEQ ID NO:190)), which cover amino acids 46 to 514 of the Cry j II proteinsequence (SEQ ID NO: 133 and 134), were created by PCR using the clonepUC19JC140iiid as a template (See Example 16). All PCR reactions werecarried out using Ultma™ DNA polymerase (Perkin Elmer Cetus, NorwalkConn.) in a 100 μl reaction. Five μl 10× Ultma™ DNA Polymerase buffer, 6μl MgCl₂ (1.5 mM final concentration), 3.2 μl 1.25 mM dNTPs (40 mM finalconcentration), and 100 pmol of each oligonucleotide in the pairsspecified below were brought to 50 μl with dH₂O. The tubes containingthese mixtures were covered with an Ampliwax Gem™ (Perkin Elmer Cetus,Norwalk Conn.) and sealed by heating to 80° C. for 5 min and thencooling to 25° C. for 1 min. Five μl 10× Ultma™ DNA Polymerase buffer, 1μg (1 μg) of DNA from clone pUC19JC140iiid, 0.5 μl of Ultma™ DNAPolymerase, and 43.5 μl dH₂O were added to every sample tube. Thesamples were then subjected to 20 rounds of amplification with aProgrammable Thermal Cycler™ (MJ Research Inc., Cambridge Mass.). Eachround of amplification consisted of heating to 94° C. for 1 min, 55° C.for 1 min, and 72° C. for 1 min. The final round of amplification wasfollowed by a 3 min incubation at 72° C.

Four sets of oligonucleotides were synthesized on an ABI 394 DNA/RNAsynthesizer (Applied Biosystems, Foster City Calif.). For construct #1,the oligonucleotides CP-38 (See Example 3) and CP-73 were used, wherebyCP-73 has the sequence 5′-GGCGGATCCTTACCATTGTTTTCCTTGCCC-3′ (SEQ IDNO:196), which is the noncoding strand sequence that corresponds tonucleotides 513-530 of FIG. 28. The nucleotides 5′-GGCGGATCC-3′ (bases1-9 of CP-73) represent a BamH I restriction site added for cloningpurposes, followed by 5′-TTA-3′ (bases 10-12 of CP-73) which encode anew stop codon. Construct #2 was generated using oligonucleotides CP-74and CP-75. CP-74 has the sequence 5′-CGGGAATTCTGGGCTGGCCAATGTAAA-3′ (SEQID NO: 197), which is the coding strand sequence that corresponds tonucleotides 531-548 of FIG. 28, and the nucleotides 5′-CGGGAATTC-3′(bases 1-9 of CP-74) represent an EcoR I restriction site added forcloning purposes. CP-75 has the sequence5′-GGCGGATCCTTATATTCCATGGCCTGGACC-3′ (SEQ ID NO: 198), which is thenoncoding strand sequence that corresponds to nucleotides 864-881 ofFIG. 28. The nucleotides 5′-GGCGGATCC-3′ (bases 1-9 of CP-75) representa BamH I restriction site added for cloning purposes, followed by5′-TTA-3′ (bases 10-12 of CP-75) which encode a new stop codon.Construct #3, was generated using oligonucleotides CP-76 and CP-77.CP-76 has the sequence 5′-CGGGAATTCAGTATAGGAAGTCTTGGG-3′ (SEQ ID NO:199), which is the coding strand sequence that corresponds tonucleotides 882-899 of FIG. 28. The nucleotides 5′-CGGGAATTC-3′ (bases1-9 of CP-76) represent an EcoR I restriction site added for cloningpurposes. CP-77 has the sequence 5′-GGCGGATCCTTAATCACTTAGCTTTATATC-3′(SEQ ID NO: 200), which is the noncoding strand sequence thatcorresponds to nucleotides 1215-1232 of FIG. 28. Nucleotides5′-GGCGGATCC-3′ (bases 1-9 of CP-77) represent a BamH I restriction siteadded for cloning purposes, followed by 5′-TTA-3′ (bases 10-12 of CP-77)which encode a new stop codon. Construct #4 was generated usingoligonucleotides CP-78 and CP-53. CP-53 is described fully in Example15, and CP-78 has the sequence 5′-CGGGAATTCATATCTTTGAAGCTTACC-3′ (SEQ IDNO: 201), which is the coding strand sequence that corresponds tonucleotides 1233-1250 of FIG. 28. Nucleotides 5′-CGGGAATTC-3′ (bases 1-9of CP-78) represent an EcoR I restriction site added for cloningpurposes.

All 4 PCRs resulted in DNA fragments of approximately 370 nucleotides inlength as visualized on ethidium bromide stained 2% agarose minigels,and all were cloned into pUC 19 as outlined in Example 16. Sequencesfrom the resultant clones were verified using the Sequenase Kit™ as inExample 16, and a single clone for each construct was chosen forsubcloning into the expression vector pET11dΔHRhis₆ (See Example 16).The clones chosen were named pUC19JC151iib, pUC19JC152iic,pUC19JC153iic, and pUC19JC154iin, for peptide constructs #1, #2, #3, and#4, respectively. DNA from each of these clones was digestedsimultaneously with EcoR I and BamH I to release the appropriate insert;these inserts were then ligated into EcoR I/BamH I digested pET11dΔHR,and the resultant clones again sequenced to verify cloning junctions.

A clone was chosen for each of the constructs #1, #2, #3, and #4, calledpET11dΔHRhis₆JC151 iib.a, pET11dΔHRhis₆JC152iic.a,pET11dΔHRhis₆JC153iic.a, and pET11dΔHRhis₆JC154iin.c, respectively, forexpression in E. coli strain BL21-DE3 as in Example 16. The fourhistidine-tagged recombinant proteins were then purified on NTA-Ni²⁺agarose, also as described in Example 16. One liter preps ofConstructs#1, #3, and #4 gave 9.3 mg, 37.4 mg, and 18.8 mg of purifiedrecombinant protein, respectively. Sequence analyses of these threerecombinant proteins verified the NH₂-terminal protein sequence, andgave an estimated purity of 67%, 95%, and 95% for Constructs #1, #3, and#4, respectively. Construct #2 was expressed at very low levels: aninitial prep of 6 L gave only about 1.5 mg of total purified proteinwith approximately 10% purity by sequence analysis. A subsequent 9 Lprep gave I mg total purified protein of 23% purity, as determined bydensitometry of a Coomassie Blue-stained SDS-PAGE gel. The isolatedprotein from these two preps was combined to give 2.5 mg protein ofapproximately 15% purity. This is referred to hereafter as #2A. A thirdlarge scale prep was prepared from a 9 L cell culture whereby theinsoluble aggregates inside the E. coli were isolated (instead of thewhole cell lysis and solubilization as above and in Example 16) by lysisof the E. coli pellet with 0.2 mg/ml lysozyme (Sigma, St. Louis Mo.) in10 ml/L culture of lysis buffer (100 mM Na₂HPO₄, 50 mM NaCl, pH8.0) for30 min on ice, followed by a rapid freeze (on dry ice/ethanol for 30min), and thaw at 37° C. The cells were then subjected to bursts ofsonication (5×20 sec) and the insoluble aggregates then collected bycentrifugation (10,000×g, 20 min). The aggregates were then washed with10 ml/L culture of the lysis buffer (without lysozyme), re-pelleted, andfinally solubilized in 10 ml/L culture 6M guanidine hydrochloride, 0.1MNa₂HPO₄, 10 mM Tris-HCl, pH 8.0. This lysate was then applied to anNTA-Ni²⁺ column and the recombinant protein purified as in Example 16.This final prep yielded 1 mg of total purified protein with a purity of40% as determined by densitometry of a Coomassie Blue-stained SDS-PAGEgel; this Construct #2 protein is referred to as #2B.

EXAMPLE 21

Identification and Development of Unique Peptides Suitable as PeptideCandidates for Use in an Injectable Multipeptide TherapeuticFormulation.

As discussed in the specification, peptides CJI-24.5 (SEQ ID NO: 129),CJI-43.39 (SEQ ID NO: 128), and CJI-44.8 (SEQ ID NO: 132) were among agroup of peptides which were “unique” as a result of modifications whichresulted in each of these peptides possessing the characteristic of“superior” solubility (i.e. stability and solubility in an aqueousbuffer of greater than 5 mg/ml over a pH range of pH6-pH8) and thecharacteristic of retaining similar T cell reactivity of the parentpeptide from which it was derived. These peptides were then tested for Tcell reactivity as discussed in Example 11 and shown in FIG. 21, whichindicated that each of peptides CJ1-24.5 (SEQ ID NO: 129), CJ1-43.39(SEQ ID NO: 128), and CJ1-44.8 (SEQ ID NO: 132) elicits T cell activityas did each of their “parent” peptides from which they were derived andthus are suitable as candidate peptides for formulating an injectabletherapeutic.

These peptides among others described earlier are “unique” in that theywere developed to fall within a very stringent set of parameters.Several different modifications of the parent peptides were attemptedprior to identifying the peptide which met all of the stringent criteriafor of a “unique” peptide which possesses “superior solubility”.

For example, the amino acid sequence of CJI-44.8 (SEQ ID NO: 132) wasderived from the protein sequence of Cry j I by first identifying thoseregions of the parent protein with high T cell reactivity using the setof overlapping peptides 20-mers as discussed in Example 6, and shown inFIG. 13, which covered the entire sequence. Two of these peptides,CJI-31 (SEQ ID NO: 54) and CJI-32 (SEQ ID NO: 56), individuallyexhibited high T-cell reactivity. Since these peptides were adjacent toeach other in the native protein sequence and overlapped by 10 residues,peptide CJI-44 was synthesized to capture the total T cell reactivity ofboth peptides. CJI-44 (SEQ ID NO: 90) (FIG. 18) is a peptide 30-merwhich contains all of the sequence present in the two 20-mers CJI-31(SEQ ID NO: 54) and CJI-32 (SEQ ID NO: 56). However, although CJI-44(SEQ ID NO: 90) posssessed T cell reactivity, when the solubility ofthis peptide was tested it had a solubility much lower than the 5 mg/mlsolubility required for a “unique” peptide.

Thus, further attempts were made increase solubility by truncation atthe N-terminus portion of CJI-44(SEQ ID NO: 90) which resulted inCJI-44.1 (SEQ ID NO: ). Additional truncation of two C-terminal residuesyielded 44.2 (SEQ ID NO: ). However, although solubility was improved inthese sequences it still did not reach the standard of “superiorsolubility”. Thus, 44.2 (SEQ ID NO: ) was further modified by theaddition of charged (hydrophilic residues) to the N-terminus and byreplacement of the hydrophobic residue Val with the less hydrophobicresidue Ala. Two of the resulting analogs, CJI-44.5 (SEQ ID NO: ) andCJI-44.6 (SEQ ID NO: ) (FIG. 20), showed increased solubility to using a“single pH point protocol procedure” (e.g. a protocol procedure whereindeterminations of solubility were made at a single pH in 100 mM sodiumphosphate buffer without mannitol under constant agitation) Twoadditional analogs were constructed in which the residue Asn wasdeleted. Of these two analogs, CJI-44.7 (SEQ ID NO: ) and CJI-44.8 (SEQID NO: 132) (FIG. 20 and FIG. 44), CJI-44.8 (SEQ ID NO: 132) was verysoluble in the “single pH point protocol” and achieved “superiorsolubility” in the “pH range protocol procedure” (i.e. whereinsolubility is measured as a function of pH in 50 mM sodium phosphatecontaining 5% mannitol with no agitation after initial mixing). CJI-44.8(SEQ ID NO: 132) was stable and soluble at greater than 5 mg/ml over thepH range pH6-pH8 in an aqueous buffer.

Peptide CJI-44.8 (SEQ ID NO: 132) was classified as a “unique” peptideafter confirmation that it retained a T-cell reactivity similar to itsparent peptides, CJI-31 (SEQ ID NO: 54), CJI-32 (SEQ ID NO: 56) andCJI-44 (SEQ ID NO: 90) (FIGS. 13 and 20). As discussed earlier uniquepeptides are particularly suitable as candidate peptides for theformulation of injectable multipeptide therapeutic compositions andformulations. Development of other “unique” peptides (e.g. CJI-24.5 (SEQID NO: 129) and CJI-43.39 (SEQ ID NO: 128)) followed a process similarto that described above for CJI-44.8 (SEQ ID NO: 132).

The combination of candidate peptides CJI-24.5 (SEQ ID NO: 129),CJI-43.39 (SEQ ID NO: 128), and CJI-44.8 (SEQ ID NO: 1-32) was tested asdescribed in earlier to determine if the combination of all threepeptides covered a sufficient percentage of T cell epitopes suitable forformulation of the peptides in a multipeptide injectable therapeuticformulation. As discussed earlier, based on an analysis of 36 patients(FIG. 45), the frequency of response at 97% represents reactivity to atleast one of the candidate peptides, indicating that this combination ofpeptides is suitable for preparation as a therapeutic composition of theinvention as well as a multipeptide formulation of the invention.

Although the invention has been described with reference to itspreferred embodiments, other embodiments, can achieve the same results.Variations and modifications to the present invention will be obvious tothose skilled in the art and it is intended to cover in the appendedclaims all such modification and equivalents and follow in the truespirit and scope of this invention.

What is claimed is:
 1. An isolated Japanese cedar pollen allergenproduced in a host cell transformed with a nucleic acid comprising thenucleotide sequence of SEQ ID NO:1, or the coding region thereof,wherein the allergen is recombinantly produced is free of other cedarpollen ailergens and is non-glycosyl.
 2. An isolated Japanese cedarpollen allergen, comprising the amino acid sequence of SEQ ID NO:2, orthe mature portion thereof, wherein the allergen is recombinantlyproduced is free of other cedar pollen allergens and isnon-glycosylated.